EP4615866A1

EP4615866A1 - Talin-based mechanosensory hydrogel

Info

Publication number: EP4615866A1
Application number: EP23817802.4A
Authority: EP
Inventors: Jennifer HISCOCK; Ben GOULT
Original assignee: University of Kent at Canterbury
Current assignee: University of Kent at Canterbury
Priority date: 2022-11-08
Filing date: 2023-11-08
Publication date: 2025-09-17
Also published as: WO2024100399A1; GB202216633D0

Abstract

Disclosed is a recombinant mechanosensory amino acid polymer comprising at least two amino acid monomer sequences and cross linking means, in which the monomers include: one or more helical structure; at least one amino acid substitution; N- and C-terminal sequence adaptations; and optional means for providing attachment to a cross-linking moiety The monomers react with the cross-linker to form the polymer. Also disclosed is a specific recombinant monomer comprising an amino acid sequence of, or derived from one or more rod domains of the protein talin which is suitable for use in the recombinant mechanosensory polymer. Also disclosed is use of the polymer and monomer as a hydrogel to absorb and/or sense kinetic energy (mechanical force) applied to the hydrogel or to protect an object from a force.

Description

MECHANOSENSORY HYDROGEL The present invention relates to protein-based materials that sense and absorb forces, such as shock from impacts applied to the material, protects objects from impacts, as well as capturing and preserving projectiles. Extreme energy dissipating materials are essential for a range of applications. The military and police force require ballistic armour to ensure the safety of their personnel, while the aerospace industry requires materials that enable the capture, preservation and study of hypervelocity projectiles. However, current industry standards display at least one inherent limitation. When impacted by a projectile, a material is exposed to a variety of phenomena simultaneously. To survive the impact, a material must contend with wave propagation (elastic, shock and plastic), fragmentation, perforation and spallation (Qiao, P.Z. et al (2008) Journal of Aerospace Engineering 21, 235-248). Thus, installing a mechanism within a material to enable effective energy dissipation is essential for multiple applications (Dyatkin, B. (2021) Mrs Bulletin 46, 201-203; Viana, J.C. (2006) Plastics Rubber and Composites 35, 260-267; Park, J.L. et al (2012) Experimental Mechanics 52, 1239-1250). Body armour is commonly used by military and civilian forces to protect the wearer against penetration from projectiles, such as bullets or shrapnel (Park, J.L. et al supra). Frequently, this armour consists of a multi-layered system, commonly a ceramic face backed by a fibre- reinforced composite (Fejdys, M. et al (2021) Journal of the Australian Ceramic Society 57, 149-161). This multi-layered design enables the hard brittle ceramic to destroy the projectile tip, in turn distributing the kinetic energy over the backing which reflects the tensile wave and captures the shattered ceramic (Reis, R.H.M. et al (2021) Polymers 13, 1203). Despite the effective penetration blocking of these armour systems, a remainder of the kinetic energy is still distributed to the wearer, often resulting in behind armour blunt trauma (Wen, Y.K. et al (2015) Journal of the Mechanical Behavior of Biomedical Materials 45, 11-21). Furthermore, during impacts, this form of armour is irreversibly damaged, compromising its structural integrity for further use. The aerospace sector utilises impact energy dissipating materials for the unique task of capture and preservation of space debris, space dust and micrometeoroids (Kearsley, A.T. (2017) 14th Hypervelocity Impact Symposium (Hvis 2017) 204, 43-50). These captured projectiles contribute towards our understanding of the local environments of aerospace equipment, including that of the international space station (Woignier, T. et al (2013) Advances in Materials Science and Engineering 484153, 1–6). Data from these experiments facilitate aerospace equipment design, improving the safety of astronauts and the longevity of costly aerospace equipment. Aerogels are the current industry standard for projectile capture and preservation, achieving energy dissipation through conversion of projectile kinetic energy into both mechanical and thermal energy (Jones, S.M. et al (2013) Icarus 226, 1-9). However, the resulting temperature elevation, further enhanced by the remarkable insulating properties of aerogels (Alwin, S. & Shajan, X.S. (2020) Materials for Renewable and Sustainable Energy 9, 1-27), can cause the aerogel structure to melt (Jones, S.M. et al supra). Furthermore, these elevated temperatures may compromise the structure of the captured projectiles, altering their chemical composition (Jones, S.M. et al supra; Bheekhun, N. et al (2013) Advances in Materials Science and Engineering 226, 1–9). This thermal and mechanical energy causes chemical bond breakage, rendering the aerogel irreversibly damaged post-impact. It is apparent from the aforementioned examples that a material utilising energy-dissipation mechanisms that re-form following the removal of force would alleviate inherent issues seen with the industry standard materials. Additionally, specifically for the aerospace sector, energy dissipation that does not result in the conversion of kinetic to thermal energy would be beneficial. To resolve these limitations the inventors have turned to nature, utilising proteins that have evolved over millennia to enable effective energy dissipation. Within the animal kingdom, proteins that offer unique mechanical properties are abundant: silk fibroin displays modifiable macroscale properties in its assembled fibre form, while elastin instils elasticity in animal tissues (Kan, A. & Joshi, N.S. (2019) Mrs Communications 9, 441-455). Although there are many proteins analogous to these examples, very few researchers have tapped into these natural resources for development of materials with novel mechanical properties (Wu, J.H. et al (2018) Nature Communications 9; Fang, J. et al (2013) Nature Communications 4, 2974, 1–10; Zhu, F.B. et al (2017) Acs Applied Materials & Interfaces 9, 11363-11367). Even fewer have tested these materials for real world applications outside of the biomedical sector (Kan, A. & Joshi, N.S. supra). Talin (talin 1 and talin 2) is the epitome of a mechanical protein, mediating the connection between the actin cytoskeleton and the integrin extracellular matrix receptors, acting as a mechanosensor. Previous work determined that, through unfolding/refolding events of its thirteen four/five helical rod domains (Bate, N. et al (2012) Plos One 7; Yao, M. et al (2016) Nature Communications 7, 11966, 1-11; Funtan, S. et al (2019) Biomimetics 4, 24, 1-18; Goult, B.T. et al (2013) J. Biological Chemistry 288, 8238-8249) when stretched within the physiologically relevant range, talin is able to maintain the average force experienced by the protein below 10 pN (Yao, M. et al supra). Furthermore, upon removal of force, refolding of the talin rod domains occurs with high fidelity over numerous force cycles (Yao, M. et al supra). The helical bundles of both talin 1 and talin 2 exhibit switch like behaviour, and act as force-dependent binary switches, opening and closing in response to changes in mechanical force. This property of helical bundles located within force transmission pathways enable mechanical signalling, but also having multiple force-dependent binary switches in series provides shock absorbing properties. Other molecules such as vinculin, catenins, filamins, Hip1R and Sla2, that are also located within force transmission pathways, contain similar helical bundles with similar properties. Talin consists of a large C-terminal rod domain that contains 13 bundles of alpha helices and an N-terminal FERM (F = 4.1 protein, E = ezrin, R = radixin, and M = moesin) domain with four subdomains: F0, F1, F2, and F3. The talin rod is comprised of 62 alpha helices that fold into 9, five-helix bundles and 4, four-helix bundles, producing 13 rod domains, R1-R13. Nine of the rod domains contains a vinculin binding site (VBS) composed of a hydrophobic surface spanning five turns of a helix. Figure 1 illustrates the boundaries of the talin 1 and talin 2 domains, and the complete amino acid sequence for mouse Talin 1 (UniProt P26039) is set out in SEQ ID NO:1: MVALSLKISIGNVVKTMQFEPSTMVYDACRMIRERIPEALAGPPNDFGLFLSD DDPKKGIWLEAGKALDYYMLRNGDTMEYRKKQRPLKIRMLDGTVKTIMVDD SKTVTDMLMTICARIGITNHDEYSLVRELMEEKKDEGTGTLRKDKTLLRDEKK MEKLKQKLHTDDELNWLDHGRTLREQGVEEHETLLLRRKFFYSDQNVDSRD PVQLNLLYVQARDDILNGSHPVSFDKACEFAGFQCQIQFGPHNEQKHKAGF LDLKDFLPKEYVKQKGERKIFQAHKNCGQMSEIEAKVRYVKLARSLKTYGVS FFLVKEKMKGKNKLVPRLLGITKECVMRVDEKTKEVIQEWSLTNIKRWAASP KSFTLDFGDYQDGYYSVQTTEGEQIAQLIAGYIDIILKKKKSKDHFGLEGDEE STMLEDSVSPKKSTVLQQQYNRVGKVEHGSVALPAIMRSGASGPENFQVG SMPPAQQQITSGQMHRGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDF ETLPPLGQDAASKAWRKNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAE TDYTAVGCAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQAAKGLAGAV SELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGESDTDPHFQDVLMQ LAKAVASAAAALVLKAKSVAQRTEDSGLQTQVIAAATQCALSTSQLVACTKV VAPTISSPVCQEQLVEAGRLVAKAVEGCVSASQAATEDGQLLRGVGAAATA VTQALNELLQHVKAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVR QARILAQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGA AAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKKLVQRLEHAAKQAAASA TQTIAAAQHAASAPKASAGPQPLLVQSCKAVAEQIPLLVQGVRGSQAQPDS PSAQLALIAASQSFLQPGGKMVAAAKASVPTIQDQASAMQLSQCAKNLGTAL AELRTAAQKAQEACGPLEMDSALSVVQNLEKDLQEIKAAARDGKLKPLPGET MEKCTQDLGNSTKAVSSAIAKLLGEIAQGNENYAGIAARDVAGGLRSLAQAA RGVAALTSDPAVQAIVLDTASDVLDKASSLIEEAKKASGHPGDPESQQRLAQ VAKAVTQALNRCVSCLPGQRDVDNALRAVGDASKRLLSDSLPPSTGTFQEA QSRLNEAAAGLNQAATELVQASRGTPQDLARASGRFGQDFSTFLEAGVEM AGQAPSQEDRAQVVSNLKGISMSSSKLLLAAKALSTDPASPNLKSQLAAAAR AVTDSINQLITMCTQQAPGQKECDNALRQLETVRELLENPVQPINDMSYFGC LDSVMENSKVLGEAMTGISQNAKNGNLPEFGDAIATASKALCGFTEAAAQAA YLVGVSDPNSQAGQQGLVEPTQFARANQAIQMACQSLGEPGCTQAQVLSA ATIVAKHTSALCNSCRLASARTANPTAKRQFVQSAKEVANSTANLVKTIKALD GDFTEENRAQCRAATAPLLEAVDNLSAFASNPEFSSVPAQISPEGRAAMEPI VISAKTMLESAGGLIQTARALAVNPRDPPRWSVLAGHSRTVSDSIKKLITSMR DKAPGQLECETAIAALNSCLRDLDQASLAAVSQQLAPREGISQEALHTQMLT AVQEISHLIEPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHP QQMALLDQTKTLAESALQLLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAV EDLTTTLNEAASAAGVVGGMVDSITQAINQLDEGPMGDPEGSFVDYQTTMV RTAKAIAVTVQEMVTKSNTSPEELGPLANQLTSDYGRLASQAKPAAVAAENE EIGAHIKHRVQELGHGCSALVTKAGALQCSPSDVYTKKELIECARRVSEKVS HVLAALQAGNRGTQACITAASAVSGIIADLDTTIMFATAGTLNREGAETFADH REGILKTAKVLVEDTKVLVQNAAGSQEKLAQAAQSSVATITRLADVVKLGAAS LGAEDPETQVVLINAVKDVAKALGDLISATKAAAGKVGDDPAVWQLKNSAKV MVTNVTSLLKTVKAVEDEATKGTRALEATTEHIRQELAVFCSPEPPAKTSTPE DFIRMTKGITMATAKAVAAGNSCRQEDVIATANLSRRAIADMLRACKEAAFH PEVAPDVRLRALHYGRECANGYLELLDHVLLTLQKPNPDLKQQLTGHSKRV AGSVTELIQAAEAMKGTEWVDPEDPTVIAENELLGAAAAIEAAAKKLEQLKPR AKPKEADESLNFEEQILEAAKSIAAATSALVKAASAAQRELVAQGKVGAIPAN ALDDGQWSQGLISAARMVAAATNNLCEAANAAVQGHASQEKLISSAKQVAA STAQLLVACKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAAAFEDQ ENETVVVKEKMVGGIAQIIAAQEEMLRKERELEEARKKLAQIRQQQYKFLPSE LRDEH (SEQ ID NO:1). Accordingly, it is against this background that the present invention has been devised. In one aspect, the present invention resides in the specific engineering of a helical bundle to impart or improve/enhance mechanosensory properties to/of the bundle. Specifically and in one aspect, the present invention encompasses a recombinant monomer comprising an amino acid sequence of, or derived from, one or more rod domains of the protein talin, wherein the monomer comprises: one or more helical structure; at least one amino acid substitution in the monomer sequence; N- and C-terminal sequence adaptations; and means to enable cross-linking of the monomer and/or means for providing attachment to a cross- linking moiety. In one embodiment, the at least one amino acid substitution is a cysteine/serine substitution if cysteine residues are present in the monomer sequence. In one embodiment, the amino acid sequence may comprise sequences from consecutive rod domains. In other words, the sequences may be consecutive as found in the wild-type sequence of talin. Examples of such sequences include rod domains 1, 2 and 3, and domains 7 and 8. In a particular embodiment, at least one of the consecutive rod domain sequences includes one or more cys/ser substitution. Ideally all the sequences in the monomer include at least one cys-serine substitution but it is not essential that substitutions are provided in all the sequences. It will be appreciated that the invention encompasses other, additional amino acid substitutions that enhance and/or improve the function of the monomer, either alone or when polymerised. For example, modification of the R3 domain itself, via substitution of buried polar residues, with a so called “IVVI” mutant, alters the mechanical properties of the domain as demonstrated previously (Yao M. et al (2014) Scientific Reports 4, 4610). In an alternative or additional embodiment, the consecutive rod domain sequences may be, or may be derived from, rod domains R1, R2 and R3. Expressed in another way, the monomer may comprise an amino acid sequence from about amino acid number 482 to about 911 of talin (talin 1) or from about amino acid number 486 to about 915 of talin (talin 2) or variations and combinations thereof. As an example, the monomer may comprise the following sequence: RGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWR KNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLT EMSRGVKLLAALLEDEGGNGRPLLQAAKGLAGAVSELLRSAQPASAEPRQN LLQAAGNVGQASGELLQQIGESDTDPHFQDVLMQLANAVASAAAALVLKAK SVAQRTEDSGLQTQVIAAATQSALSTSQLVASTKVVAPTISSPVSQEQLVEA GRLVAKAVEGSVSASQAATEDGQLLRGVGAAATAVTQALNELLQHVKAHAT GAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKA DAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREA AEGLRMATNAAAQNAIKKGT (SEQ ID NO:2). In an alternative embodiment, the monomer may comprise at least two (two or more) repeats of an amino acid sequence of, or derived from, a single rod domain. For example, the single rod domain may be, or may be derived from, rod domain R1, rod domain R3, rod domain R9, or rod domain R13. The upper limit of the number of sequence repeats will be determined by the particular sequence used, the ability of the monomer to form a functional molecule, as well as having the desired function and properties, and the ability to produce the monomer cost effectively and accurately. For example, wild type talin has thirteen rod domains but a recombinant monomer including up to at least twenty domains is conceivable and contemplated. In an alternative embodiment, the monomer may comprise at least two (two or more) repeats of an amino acid sequence of, or derived from, non-contiguous rod domains. For example, the monomer may be, or may be derived from rod domains R3 and R9 and the sequences ordered in the monomer as R3-R9-R3-R9, R3-R9-R3 or R9-R3-R9. In a particular embodiment, at least one of the rod domain sequences includes one or more cys/ser substitution. As above, ideally all the sequences in the monomer include at least one cys-serine substitution, where cysteine residues are present in the sequence, but it is not essential that substitutions are provided in all the sequences. It will be appreciated that the invention encompasses other, additional amino acid substitutions that enhance and/or improve the function of the monomer, either alone or when polymerised. In a specific embodiment, the R1 rod domain may have the following sequence: RGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDE SKHEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALL EDEGGNGRPLLQAAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQI (SEQ ID NO:3). In another specific embodiment, the R3 rod domain may have the following sequence: AHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVN AIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRL REAAEGLRMATNAAAQNAIKK (SEQ ID NO:4). In another specific embodiment, the R9 rod domain may have the following sequence: APGQLESETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAV QEISHLIEPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQ MALLDQTKTLAESALQLLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAVED LTTTLNEAASAA (SEQ ID NO:5). In yet another specific embodiment, the R13 rod domain may have the following sequence: DPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQILEAAKSIAAATSALV KAASAAQRELVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNNLSEAANAAVQG HASQEKLISSAKQVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRASDNLVKAA QKAAAFED (SEQ ID NO:6). In an additional or alternative embodiment, the repeats of the single (sequential or non- sequential) rod domain sequences may be separated by a linker. An example of a suitable linker is glycine serine linker such as [GGGGGS]x [SEQ ID NO:7], where x = one or more repeats. In a particular example, the linker sequence is GGGGGSGGGGGS (SEQ ID NO:8). While glycine serine linkers are particularly suitable (see van Rosmalen et al (2017) Biochemistry 56, 50, 6565-6574), it will be appreciated that any suitable linker may be used. The skilled person knows that a particularly suitable linker is a short one that has sufficient flexibility and provides enough distance between the sequences, so the domains are not sterically hindered. It will also be appreciated that the maximum number of repeats of a linker sequence is also easily derivable and depends on the length of the linker sequence and the distance required between domains. In a particular embodiment, at least one of the rod domain sequences includes one or more cys/ser substitution. As above, ideally all the sequences in the monomer include at least one cys-serine substitution, where cysteine residues are present in the sequence, but it is not essential that substitutions are provided in all the sequences. It will be appreciated that the invention encompasses other, additional amino acid substitutions that enhance and/or improve the function of the monomer, either alone or when polymerised. In a further embodiment, the N-terminal of the monomer may include an adaptation having the sequence [GGGGS]_xGS (SEQ ID NO:9), where _x = one or more repeats. This sequence acts as a flexible linker, the function of which is to remove the protein monomer bulk away from any cross-linker reaction site, assisting the reaction of a single cross-linker with multiple protein monomers. While it will be appreciated that any equivalent and/or suitable sequence may be used, a particular example of such a sequence may be CGGGGSGGGGSGGGGSGS (SEQ ID NO:10). In a yet further embodiment, the C-terminal may include an adaptation having the sequence [GGGGS]_x (SEQ ID NO:11), where _x = one or more repeats. As with the N-terminal adaptation sequence, the C-terminal adaptation also acts as a flexible linker and functions in the same way as the N-terminal adaptation sequence. In a particular example, the adaptation sequence may be GGGGSGGGGSGGGGSC (SEQ ID NO:12). It will be appreciated that any suitable adaption sequence(s) may be used and that SEQ ID NOs:11 and 12 are merely non-limiting examples of a flexible linker. The means to enable cross-linking of the monomer and/or for providing attachment to a cross-linking moiety may be any suitable means, such as an amino acid, an amino acid sequence, or a chemical entity, that enables one monomer to be attached or cross-linked to at least one other. For example, the amino acid sequence may include a cysteine residue at the leading end of one or each of the terminal adaptor sequences allow the monomer to be attached to another moiety, such as a linker or cross-linking moiety. During polymerisation, the cysteine residue reacts with a cross-linking moiety added to the monomer sequence in its place. The terminal cysteine covalently couples the monomer sequence to other moieties. It will be appreciated that the invention encompasses monomer sequences that include other or additional entities that enable the cross-linking of monomers, for example protein interaction motifs such as SpyTag/SpyCatcher and AviTag^TM, as well as the inclusion of binding sites for other proteins and sequences that impart selected characteristics to the monomer that encourage, promote or enable cross-linking. An example of means to encourage, promote or enable cross-linking includes non-covalent cross-linking via one or more amino acid sequences as part of the monomer sequence, in which the additional sequence imparts characteristics to the monomer that encourage, promote or enable cross-linking. For example, additional sequences may be in the form of a coiled coil. According to Uniprot, coiled coils are built by two or more alpha helices that wind around each other to form a supercoil. There can be two, three or four helices in the bundle and they might either run in the same (parallel) or in the opposite (antiparallel) directions. Sequences in these coils impart a hydrophobic side or region and a hydrophilic side or region to the three-dimensional structure such that, in an aqueous environment, the hydrophobic sides interact with each other, shielding them from the aqueous polar environment. Thus, the non-covalent hydrophobic/hydrophilic effect may be used to drive self-association of the residues which, when attached to the terminal ends of the protein monomeric units will instigate cross-linking. In another embodiment, the amino acid sequence of the monomer may further comprise an expression sequence. It will be appreciated that the expression sequence will be designed to facilitate expression of the monomer sequence in a particular expression system and is typically cleaved from the monomer sequence during expression. Therefore, while any suitable expression sequence or affinity tag may be used to enable the purification of the protein, an example of a particular expression sequence is: MHHHHHHGKPIPNPLLGLDSTENLYFQ (SEQ ID NO:13). An additional or alternative expression sequence is MHHHHHHENLYFQG (SEQ ID NO:14). A yet further additional or alternative expression sequence is MHHHHHHGCGGGGSGGGGSGGGGSGS (SEQ ID NO:15) where the His-tag is not cleavable. Examples of monomer sequences are: BOLD = adaptions. Italic = expression tagged removed in production. C = cysteine attachment sites for attachment of the crosslinker. pGEL01 – engineered expression sequence of R1-R2-R3 rod domains in series: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSRGHMPPL TSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHSQVD AITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQ AAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGESDTDPHFQDVLMQ LANAVASAAAALVLKAKSVAQRTEDSGLQTQVIAAATQSALSTSQLVASTKVVAPTISSPVS QEQLVEAGRLVAKAVEGSVSASQAATEDGQLLRGVGAAATAVTQALNELLQHVKAHATGA GPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSR KLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGTGG GGSGGGGSGGGGSC (SEQ ID NO:16). pGEL02 sequence – engineered expression sequence of 3 copies of the R9 rod domain in series: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAPGQLES ETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEA SQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGG NPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGGGGSAPGQLESETAIA ALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEASQLG HKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGGNPKQ AAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGGGGSAPGQLESETAIAALNS SLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEASQLGHKVS QMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGGNPKQAAHT QEALEEAVQMMTEAVEDLTTTLNEAASAAGGTGGGGSGGGGSGGGGSC (SEQ ID NO:17). pGEL03 sequence – engineered expression sequence of 3 copies of the R3 rod domain in series: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAHATGAG PAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRK LLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGGGG SGGGGSAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIK ADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMAT NAAAQNAIKKGGGGSGGGGSAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQ ARILAQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQ QRLREAAEGLRMATNAAAQNAIKKGTGGGGSGGGGSGGGGSC (SEQ ID NO:18). pGEL04 sequence – engineered expression sequence of 3 copies of the R1 rod domain in series: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSRGHMPPL TSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHSQVD AITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQ AAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGGGGSGGGGSRGHM PPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHS QVDAITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGR PLLQAAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGGGGSGGGGS RGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESK HEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEG GNGRPLLQAAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGTGGGG SGGGGSGGGGSC (SEQ ID NO:19). pGEL05 sequence – engineered expression sequence of R9-R3-R9 rod domains in series: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAPGQLES ETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEA SQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGG NPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGGGGSAHATGAGPAG RYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRKLLS AAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGGGGSGG GGSAPGQLESETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLI EPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQ LLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGTGGGGSGGGG SGGGGSC (SEQ ID NO:20). pGEL06 sequence – engineered expression sequence of R9-R3-R9 rod domains in series: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAHATGAG PAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRK LLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGGGG SGGGGSAPGQLESETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEI SHLIEPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAE SALQLLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGG GGSAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADA EGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAA AQNAIKKGTGGGGSGGGGSGGGGSC (SEQ ID NO:21). pGEL07 sequence – engineered expression sequence of 3 copies of the R13 rod domain in series MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSDPTVIAE NELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQILEAAKSIAAATSALVKAASAAQRE LVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNNLSEAANAAVQGHASQEKLISSAK QVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAAAFEDGGGGSG GGGSDPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQILEAAKSIAAATSA LVKAASAAQRELVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNNLSEAANAAVQGH ASQEKLISSAKQVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAA AFEDGGGGGSGGGGSDPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQI LEAAKSIAAATSALVKAASAAQRELVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNN LSEAANAAVQGHASQEKLISSAKQVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRA SDNLVKAAQKAAAFEDGGGGSGGGGSGGGGSC (SEQ ID NO:22). It is to be understood that the present invention also encompasses nucleotide sequences and expression systems comprising these nucleotide sequences that are suitable for expressing the amino acid sequences described herein above. In second aspect, the present invention resides in a recombinant mechanosensory amino acid polymer in which the polymer comprises at least two amino acid monomer sequences and cross-linking means, characterised in that the monomer sequences include: one or more helical structures; at least one amino acid substitution; N- and C-terminal sequence adaptations; and, means to enable cross-linking of the monomers and/or means for providing attachment to a cross-linker, wherein the monomers are cross-linked to form the polymer. In an embodiment, the mechanosensory polymer of the invention may comprise monomers derived from a single protein or monomers derived from related or different proteins. It will be appreciated that the at least two monomers may be selected to impart particular properties to the polymer. In a particular example, the polymer may be designed to absorb and/or sense kinetic energy (i.e. a shock) from a force such as a mechanical impact. As a result, monomers may be selected based on their ability to impart these capabilities to the polymer. Examples of mechanosensory proteins include vinculin, Sla2, Hip1R, filamin, and catenins. In a particular embodiment and without wishing to be bound by theory, it is believed that a helical structure, such as the helical bundles found in the protein talin, opens and closes in response to kinetic energy, such as provided by a mechanical force, thereby acting as a force-dependent “switch” domain. Therefore, in some embodiments the switch domains may open in response to kinetic energy (force) and remain open. In other embodiments, the domains may refold and effectively close. In this way, the former provides a polymer for a single use while the latter provides a polymer for multiple uses that is able to absorb (dissipate) and/or sense shock multiple times. In another embodiment, the switch domains may be selected to respond to different amounts of force (kinetic energy) to give the polymer the ability to absorb and/or sense shock in a range, or selection of ranges, of forces. Alternatively or in addition, multiple switch domains may be activatable at different levels of force. For example, a particular force may cause the opening or unfolding of one or more helical structures while a greater or different force recruits the unfolding of additional or other helical structures. As a result, the polymer of the invention may be “tuneable” according to the amount of force absorbance and dissipation required. Examples of suitable amino acid monomers include those derived from the protein talin, such as a monomer derived from one or more rod domains of talin (talin 1 and /or talin 2) as described herein above, vinculin, Sla2, Hip1R, filamin, and catenins. In a particular embodiment, all of the cysteine residues in the monomers are substituted with serine where native cysteine residues are present. As above, ideally all the sequences in each monomer that include native cysteine residues include at least one cys-serine substitution, but it is not essential that substitutions are provided in all the monomers or sequences of each monomer. In another embodiment, the N-terminal of each monomer includes an adaptation having the sequence [GGGGS]xGS (SEQ ID NO:9), where x = one or more repeats. As described above, the adaptation sequence acts as a flexible linker to position the monomer structure in such a way as to facilitate and/or maximise the ability of the cross-linking moiety to react with multiple monomers. In a particular non-limiting example, the N-terminal adaptation may comprise the sequence CGGGGSGGGGSGGGGSGS (SEQ ID NO:10). In a yet further embodiment, the C-terminal of each monomer may include an adaptation having the sequence [GGGGS]x (SEQ ID NO:11), where x = one or more repeats. Again, this adaptation sequence acts as a flexible linker in the same was as the N-terminal adaptation sequences described herein and above. In a particular non-limiting example, the C terminal adaptation may comprise the sequence GGGGSGGGGSGGGGSC (SEQ ID NO:12). As above, it will be appreciated that any suitable adaption sequence(s) performing the same function may be used and that the sequences set out hereinabove are purely for illustrative purposes. As explained herein and above, the means to enable cross-linking of the monomers and/or for providing attachment to a cross-linker may be any suitable means, such as an amino acid, an amino acid sequence, or a chemical entity, that enables one monomer to be attached to at least one other. A particular example of such means is cysteines at a or the leading end of the terminal adaptor sequences. During polymerisation of the monomers, a linker or crosslinking moiety is attached to the monomer sequence via reaction with cysteine. It will be appreciated that the invention encompasses alternative or additional entities that enable the cross-linking of monomers, for example protein interaction motifs such as SpyTag/SpyCatcher and AviTag^TM, as well as the inclusion of binding sites for other proteins and/or, as described above, sequences that impart selected characteristics to the monomers that encourage, promote or enable non-covalent cross-linking, such as coiled coils. It In Formula I In a yet further embodiment, the polymer may further comprise a marker or binding entity which changes the properties of the polymer on unfolding and/or refolding of the helical structure. In other words, the marker or binding entity acts as a sensory marker to indicate when a change has occurred. The marker or binding entity may impart a fluorescence or a change in colour, luminescence, or opacity on either unfolding or refolding of the helical structure. It will be appreciated that the invention encompasses the embodiment where a dimensional or conformational change in the polymer structure that occurs as a result of unfolding/refolding causes the marker or binding entity to impart a colour change or fluorescence, as well as the embodiment where unfolding/refolding of the helical structures allows the binding or unbinding of the marker or binding entity to the helical structure, which interaction causes a fluorescence or a change in colour, luminescence, or opacity. The invention also encompasses markers and binding entities that specifically bind to the closed (folded) state of the helical structures, i.e. when the structures are in a state of low tension, or the open (unfolded) structures, i.e. when the structures are under tension or compression. In an embodiment, the marker or binding entity may impart a reversible or non-reversible change to the polymer when the helical structure is in an unfolded state, i.e. when the structure is under tension or compression. Examples of suitable markers or binding entities include vinculin and a marker that includes one or more leucine-aspartic acid (LD) motifs, such as LD motifs derived from KANK1-4, Deleted in Liver Cancer 1 (DLC1) and Rap1-GTP-interacting adaptor molecule (RIAM) proteins, and aptamers, intrabodies or antibodies raised against specific domain states. In a yet further aspect, the present invention resides in the use of mechanosensory hydrogel comprising a recombinant mechanosensory monomer or polymer as described herein to absorb, dissipate and/or sense force applied to the hydrogel. Expressed in another way, the present invention also resides in a method of absorbing and/or sensing force, particularly a force or kinetic energy applied to a surface, wherein the force or energy is absorbed, dissipated and/or sensed by a mechanosensory hydrogel (applied to the surface) comprising a recombinant mechanosensory monomer or polymer as described herein. The present invention also resides in the use of a mechanosensory hydrogel comprising a recombinant mechanosensory monomer or polymer as described herein to protect an object from a force or impact by or with another object. Such protective use finds particular application in the protection of space equipment against micro impacts from the likes of micrometeoroids and orbital and space debris (MMOD). According to NASA, space debris encompasses both natural meteoroid and artificial (human-made) orbital debris. It will be appreciated that such use also encompasses a method of protecting an object from a force or impact by or with another external object by the application or inclusion of a mechanosensory hydrogel comprising a recombinant mechanosensory monomer or polymer as described herein. The mechanosensory hydrogel comprising a recombinant mechanosensory monomer or polymer as described herein finds a yet further use as an adhesive and/or coating of materials used in products designed to protect an object or person from a force or impact from or with another object. Expressed in another way, also disclosed herein is a method of adhering or coating materials used in products designed to protect an object or person from a force or impact from or with another object by coating or incorporating a mechanosensory hydrogel comprising a recombinant mechanosensory monomer or polymer as described herein. In a particular embodiment, the force may be a mechanical shock or shock wave resulting from impact with an object, for example a high velocity, hypervelocity and/or explosive object such as a projectile, a bullet or space debris. In a further embodiment, the mechanosensory hydrogel additionally substantially retains the object within its structure. The present invention will now be described in more details with reference to the following non-limiting examples and figures in which: Figure 1. Domain map of the boundaries of the talin 1 and talin 2 domains. Figure 2. Chemical structures of compounds 1 and 2. a. Compound 1 is a control compound and b. compound 2 is a tripodal crosslinker. Figure 3.¹H NMR spectra of compound 1 in DMSO-d6 conducted at 298.15 K. Figure 4.¹H NMR spectra of compound 2 in DMSO-d6 conducted at 298.15 K. Figure 5. Liquid chromatography-mass spectrometry characterisation of compound 1 and 2. a. Kank1 peptide (95% purity) (0.10 mM, 4067.01 g/mol) used for crosslinking characterisation. b. Compound 1 (1.00 mM, 153.08 g/mol) bound to single Kank1 peptide (0.10 mM, 4067.01 g/mol) confirming the maleimide group was capable of binding biological macromolecules. c. Compound 2 (1.00 mM, 386.12 g/mol) acting as a crosslinking agent bound to three Kank1 peptides (1.00 mM) confirming all maleimide groups were capable of binding biological macromolecules. Figure 6. Expression and purification of ¹⁵N-labelled monomers. a. SDS-PAGE gels from Ni affinity and anion exchange purification for pGEL01. b. SDS-PAGE gels from Ni affinity and anion exchange purification for pGEL02. c. SDS-PAGE gels from Ni affinity and anion exchange purification for pGEL03. d. SDS-PAGE gels from Ni affinity and anion exchange purification for pGEL04. e. SDS-PAGE gels from Ni affinity and anion exchange purification for pGEL05. Red arrows show purified monomers. f. SDS-PAGE gels from Ni affinity and anion exchange purification for pGEL06. Red arrows show purified monomers. Figure 7. Comparative expression time course of pGEL01 in different growth media and conditions, following induction of gene expression. a. BL21(DE3) pGEL01 expression in LB medium at 18°C. b. BL21(DE3) pGEL01 expression in TB medium at 37°C. c. BL21(DE3) pGEL01 expression in TB medium at 25°C. Cell fractions standardised to 10 OD units. Arrows indicate pGEL01 protein. S = soluble, In =insoluble. Figure 8. Design of pGEL01 monomers including Spy peptides. a. SpyCatcher-pGEL01. SpyCatcher peptides circled in green. b. SpyTag-pGEL01. SpyTag peptides circled in purple. c. AlphaFold predicted 3D binding structure of SpyCatcher-pGEL01 monomers with SpyTag-pGEL01 monomers. Figure 9. Expression and purification of pGEL01 monomers with Spy peptides. SDS-PAGE gels of nickel affinity chromatography samples from purification of a. SpyCatcher-pGEL01. b. SpyTag-pGEL01. Red arrows show purified monomers. Figure 10. Circular Dichroism showing a. Alpha helical folding was retained in pGEL01 compared to the wild type R1-R3 domains of talin1 following one mutation in R1 and four mutations in R2. b. Only small changes in Tm between the two proteins were observed. Figure 11. Circular Dichroism showing a. Alpha helical folding is retained in the mutated R2 following four cysteines mutated to serines. b. A change in Tm is seen between the mutated R2 and wild type R2, suggesting minor alterations to stability has occurred. Figure 12. NMR spectra of pGEL01 overlaid with the single talin R3 domain. a. NMR spectrum of pGEL01 alone and b. pGEL01 (blue) overlaid with mouse talin1 R3 (red). Figure 13. NMR spectra of pGEL02 and pGEL03 overlaid with the corresponding single talin rod domains. a. NMR spectrum of pGEL02 alone and b. pGEL02 (red) overlaid with mouse talin1 R9 (sky blue). c. NMR spectrum of pGEL03 alone and d. pGEL03 (magenta) overlaid with mouse talin1 R3 (blue). Figure 14. NMR spectra of pGEL04 overlaid with the corresponding single talin rod domains. a. NMR spectrum of pGEL04 alone and b. pGEL04 (red) overlaid with mouse talin1 R1 (sky blue). Figure 15. NMR spectrum of a. pGEL05 and b. pGEL06. pGEL05 spectrum (blue) overlaid with R3 (magenta; c) and with R9 (cyan; d), and e. enlargement of pGEL05 spectrum (blue) with R3 (magenta) and R9 (cyan). The outline of the enlarged region in e. is shown in c. and d. with a red square. Figure 16. Circular dichroism (CD) for pGEL02, pGEL03, pGEL05 and pGEL06. Melting curves taken at 222 nm (upper) and UV scans (lower) of a. pGEL02, b. pGEL03, c. pGEL05, and d. pGEL06. Melting curves were collected at 222 nm from 20°C to 90°C for pGEL02 and pGEL03, and from 20°C to 95°C for pGEL05 and pGEL06. UV scans were carried out at 20°C and 90°C for pGEL02 and pGEL03, and at 20°C and 95°C for pGEL05 and pGEL06. Figure 17. Circular dichroism (CD) for pGEL01. Melting curves taken at a.222 nm and b. CD scans on pGEL01. Melting curves and CD scans were both collected from 20°C to 95°C. Three stages of melting (Tm) were identified at 63.3, 68.5 and >75°C. Figure 18. Circular dichroism (CD) for pGEL04. Melting curves taken at a.222 nm and b. CD scans on pGEL04. Melting curves at and UV scans were both collected from 20°C to 95°C. The melting temperature (Tm) was identified at 68.0°C. Figure 19. Melting curve of mouse talin1 R13 at 222 nm from 20°C to 90°C. Figure 20. HSQC spectra of ¹⁵N-labelled mutated R1 with wild type R1 chemical shift assignments from Banno et al. overlaid. Figure 21. HSQC spectra of ¹⁵N-labelled mutated R1 with wild type R1 assignments from Banno et al. overlaid, centred on the region where the cysteine in wild type R1 is positioned. Figure 22. Resistance of 300 mg/mL TSAM to shear strain at 25°C. Five consecutive amplitude sweeps using Anton Parr rheometer were performed on the same piece of 700 μL TSAM gel (pGEL01), the amplitude range increased from 0.01 – 100 γ shear strain per sweep. A 2-minute rest was set to occur between each sweep. a. First sweep. b. Second sweep. c. Third sweep. d. Fourth sweep. e. Fifth sweep. G’ (blue) = storage modulus; G” (red) = loss modulus. Figure 23. The design concept of a talin shock absorbing material (TSAM). a. compounds 1 and 2, b. The pGEL01 monomer in the folded state, green boxes = flexible linkers, blue box = mutated R1-R3 domains of talin. c. Resulting gelation for each compound. d. Representation of the three-armed network structure formed from compound 2 with no applied strain. e. pGEL01 in fully folded state presents length of ≈15 nm. f. When exposed to strain pGEL01 unfolds into a linear string of helices extending to ≈65 nm in length. g. When exposed to higher strain, pGEL01 unfolds fully into extended polypeptide, increasing to a length of ≈156 nm. Complete refolding can occur once strain is removed. h. Representation of the three-armed network structure with applied strain, causing extension of protein into opened helices form, increasing fibre length. Figure 24. The internal fibre structure of TSAM and its macroscale characterisation. a. Immunogold-stained TSAM imaged with transmission electron microscopy (TEM) showing lattice structure of connected pGEL01 proteins. Gold particles are observed as black dots, some of which are highlighted with red arrows (Scale bar = 200 nm). b. The dense fibre structure of TSAM displaying a porous network imaged with scanning electron microscopy (SEM) on secondary electron mode (Scale bar = 50 µm). Pore sizes are on the range of a few µm. c. Energy Dispersive X-Ray (EDX) analysis of SEM image in b. sulphur = yellow, carbon = red, oxygen = green, sodium = teal, phosphorus = purple. (Scale bar = 50 µm) d. TSAM slightly stretched. e. TSAM stretched to 3x its length. f-h: Rheological measurements of TSAM. f. G’ as a product of shear strain (error bars = SEM) for sweeps 1 (squares), 2 (circles), 3 (triangles), 4 (inverted triangles) and 5 (diamonds). g. Phase angle against shear strain for sweeps 1-5 on TSAM. h. Shear stress against shear strain for sweeps 1-5 on TSAM. i. Schematic summary of the events hypothesised to occur over 5x repeated oscillatory sweeps. Figure 25. a. Amplitude sweep 1 on TSAM (error bars = SEM). G’ and G” against shear strain and shear stress against shear strain; b. Amplitude sweep 2 on TSAM (error bars = SEM). G’ and G” against shear strain and shear stress against shear strain; c. Amplitude sweep 3 on TSAM (error bars = SEM). G’ and G” against shear strain and shear stress against shear strain; d. Amplitude sweep 4 on TSAM (error bars = SEM). G’ and G” against shear strain and shear stress against shear strain; e. Amplitude sweep 5 on TSAM (error bars = SEM). G’ and G” against shear strain and shear stress against shear strain. Figure 26. Resistance of 200 mg/mL TSAM (pGEL01) to shear strain at different temperatures. Five consecutive amplitude sweeps using the Anton Parr rheometer were performed per temperature on the same piece of 700 μL TSAM gel, the amplitude range increased from 0.01 – 100 γ shear strain per sweep. A wait condition was set until the new temperature was reached before the first sweep was performed, and a 2-minute rest was set to occur between each sweep. a. First sweep at 10°C. b. Fifth sweep at 10°C. c. First sweep at 25°C. d. Fifth sweep at 25°C. e. First sweep at 35°C. f. Fifth sweep at 35°C. g. First sweep at 45°C. h. Fifth sweep at 45°C. i. First sweep at 65°C. j. Fifth sweep at 65°C. k. First sweep at 85°C. l. Fifth sweep at 85°C. G’ (blue) = storage modulus; G” (red) = loss modulus. Figure 27. Optical density scan of 200 mg/mL TSAM (pGEL01) between 300 - 1000 nm wavelengths at different incubation temperatures. Figure 28. FTIR peak deconvolution of 200 mg/mL TSAM (pGEL01) following heating. a. Pre-crosslinker, 25°C. b. Post-crosslinker, 25°C. c. Post-crosslinker, 35°C. d. Post- crosslinker, 45°C. e. Post-crosslinker, 85°C. Figure 29. Gel filtration results confirming green fluorescent protein tagged-vinculin domain 1 protein (GFP-VD1) binds to pGEL01. GFP-VD1 = dotted black line, pGEL01 = dotted red line, GFP-VD1 + pGEL01 at 1:1 = solid black line, GFP-VD1 + pGEL01 at 3:1 = solid red line. Figure 30. a. Amplitude sweeps from rheological characterisation of TSAM after treatment with phosphate buffer (black), GFP (green) or GFP-VD1 (blue). G’ and G” against shear strain and shear stress against shear strain for a. sweep 1; b. sweep 2; c. sweep 3; d. sweep 4; e. sweep 5. Figure 31. Effects of GFP-VD1 on TSAM. a. Representation of GFP-VD1 binding to unfolded pGEL in TSAM fibres, with resulting cartoon protein figures created in PyMOL using VD1 PDB structure 1U6H (Fillingham, I. et al (2005) Structure 13, 65-74). b. Shear stress as a product of shear strain for buffer (triangles), GFP-VD1 (squares) and GFP (circles), showing GFP-VD1 treated TSAM reaches its yield point between 46-68% shear strain. c. Transmitted light image of GFP-VD1 localised to TSAM fibres (scale bar = 20 µm). d. Maximum projection widefield fluorescent image of c. (scale bar = 20 µm) with fibres showing localised GFP-VD1 indicated by arrows. e. Transmitted light image of GFP in TSAM (scale bar = 20 µm). f. Maximum projection widefield fluorescent image of e. showing GFP sitting in void space, with fibres that were previously visible as bright green in d. this time visible as darker structures indicated by arrows (Scale bar = 20 µm). Figure 32. Hypervelocity impact study on TSAM. a. SEM image (Scale bar = 60 µm) of a basalt particle used as the projectile and representation of how the basalt is loaded into a sabot and its release during a shot. b. diagram of the light gas gun apparatus with the key stages after the shot is triggered. c. image of TSAM and how it is prepared as a target. The TSAM is loaded into a target plate constructed of steel (Blast tank exit aperture (BTEA), stainless 304), with tape used to seal the back of the hole, followed by an aluminium back plate (Al 5083). d-e. results from control gel d. Destroyed control gel after basalt impact at 1.5 km/s. e. Hole formed in tape from basalt projectile. f. Crater formed in aluminium back plate. g-I: results from TSAM g. Mostly intact TSAM after basalt impact at 1.5 km/s. h. Tape with no hole, containing several caught basalt particles in the transparent TSAM attached to its surface. i. undamaged aluminium back plate. j. SEM image of intact basalt particle caught by TSAM after impact at 1.5 km/s. (scale bar = 45 µm) k. SEM image of another basalt particle caught by TSAM after impact at 1.5 km/s. (scale bar = 30 µm) l. SEM image of a fragment of the aluminium (Al 7075) burst disc that impacted TSAM during the 1.5 km/s basalt shot (scale bar = 50 µm). Figure 33. Images from light gas gun (LGG) experiment. a. Intact TSAM attached to tape on the back of the BTEA. b. Image under light microscope of basalt particles integrated into the TSAM shown in a. c. Resulting dent on the back of the aluminium back plate from the control LGG shot. d. side by side comparison of the control LGG shot back plate and TSAM LGG shot. Figure 34. SEM images of basalt particles and corresponding elemental dispersive X-ray analysis. a. Basalt particle before being shot from a LGG and its corresponding EDX analysis. b. Basalt particle 1 and its corresponding EDX analysis. c. Basalt particle 2 and its corresponding EDX analysis. Figure 35. SEM images of burst disk aluminium fragment (a) and corresponding elemental dispersive X-ray analysis confirming aluminium (b). Figure 36. Testing of the adhesive effects of a TSAM with armour materials. a. Aramid fibres. b. TSAM adhered to aramid fibres. c. TSAM adhered to aluminium oxide ceramic beads. d. Breakage of ceramic surface when force applied to TSAM-adhered ceramic beads. Figure 37. SEM imaging of TSAM adhered to aramid fibres. a. SEM image of aramid fibres embedded within TSAM. b. Elemental X-ray analysis of aramid fibres bonded by TSAM. Key: Dark blue: Phosphorous. Light blue: Oxygen. Red: Carbon. Green: Sulphur. Figure 38. SEM imaging of TSAM adhered to aluminium oxide ceramic, following the separating of two beads. a. SEM image of the surface of a ceramic bead adhered with TSAM. Light areas indicate ceramic material; dark areas indicate TSAM. b. SEM image of the surface of a ceramic bead adhered with TSAM following separation. SEQ ID NO:1 – complete amino acid sequence for mouse Talin 1 (UniProt P26039). SEQ ID NO:2 – engineered amino acid sequence of rod domains R1-R2-R3 of Talin 1. SEQ ID NO:3 – engineered amino acid sequence of the R1 rod domain derived from mouse Talin 1. SEQ ID NO:4 – engineered amino acid sequence of the R3 rod domain derived from mouse Talin 1. SEQ ID NO:5 – engineered amino acid sequence of the R9 rod domain derived from mouse Talin 1. SEQ ID NO:6 – engineered amino acid sequence of the R13 rod domain derived from mouse Talin 1. SEQ ID NO:7 – linker sequence for use between single (non-sequential) rod domain sequences. SEQ ID NO:8 – specific inter-sequence linker sequence. SEQ ID NO:9 – N-terminal adaptation. SEQ ID NO:10 – specific N-terminal adaptation. SEQ ID NO:11 – C-terminal adaptation. SEQ ID NO:12 – specific C-terminal adaptation. SEQ ID NO:13 – example expression sequence. SEQ ID NO:14 – example expression sequence. SEQ ID NO:15 - example expression sequence. SEQ ID NO:16: – pGEL01: engineered expression sequence of R1-R2-R3 rod domains in series. SEQ ID NO:17 – pGEL02: engineered expression sequence of three copies of the R9 rod domain sequence in series. SEQ ID NO:18 – pGEL03: engineered expression sequence of three copies of the R3 rod domain sequence in series. SEQ ID NO:19 – pGEL04: engineered expression sequence of three copies of the R1 rod domain sequence in series. SEQ ID NO:20 – pGEL05: engineered expression sequence of R9-R3-R9 rod domain sequences in series. SEQ ID NO:21 – pGEL06: engineered expression sequence of R3-R9-R3 rod domain sequences in series. SEQ ID NO:22 – pGEL07: engineered expression sequence of three copies of the R13 rod domain sequence in series. SEQ ID NO:23 – Sequence for wild-type R1 derived from mouse talin 1 and including an expression sequence. SEQ ID NO:24 – Sequence for mutated R1 derived from mouse talin 1 and including an expression sequence. SEQ ID NO:25 – Sequence for wild-type R2 derived from mouse talin 1 and including an expression sequence. SEQ ID NO:26 – Sequence for mutated R2 derived from mouse talin 1 and including an expression sequence. SEQ ID NO:27 – Sequence for wild-type R3 derived from mouse talin 1 and including an expression sequence. SEQ ID NO:28 – Sequence of green fluorescent protein including an expression sequence. SEQ ID NO:29 – Sequence of GFP-labelled vinculin VD1 including an expression sequence. Cross-linking moieties Compound 1 (control): Compound 1 (1-butyl-1H-pyrrole-2,5-dione; Figure 2a) was synthesised as described by Eloh et al ((2016) J. Agricultural and Food Chemistry 64, 4876-4881) with minor modifications. Maleic anhydride (1.00 g, 10.00 mmol) was dissolved in dichloromethane (DCM) (15.00 mL). N-butylamine (1.00 mL, 1.00 mmol) was added and the mixture was stirred at room temperature for 1 hour. The solvent was removed in vacuo and the resulting white powder was re-dissolved in acetic anhydride (6.00 mL). To this solution, sodium acetate (0.50 g, 6.10 mmol) was added, and the mixture was heated at 80 ^C under reflux for 2 hours. The solution was diluted with distilled water (50.00 mL) and washed with diethyl ether (3 x 50.00 mL). The organic layer was collected and further washed with 0.1 M hydrochloric acid (1 x 50.00 mL) and 0.1 M sodium hydroxide (1 x 50.00 mL). The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated in vacuo to give the crude product as a colourless liquid. The N-butylmaleimide was further purified using silica chromatography, 85:15 (ethyl acetate:hexane), producing a yellow oil with a yield of 11% (0.17g, 11.00 mM).¹H NMR (400 MHz, 298 K, DMSO-d₆): δ: 7.01 (s, 2H), 3.38 (t, J = 7.06 Hz, 2H), 1.46 (m, 2H), 1.21 (m, 2H), 0.86 (t, J = 7.36 Hz, 3H). NMR consistent with previously reported values (see Figure 3). Compound 2 (Cross-linker): Compound 2 (1,1',1''-(nitrilotris(ethane-2,1-diyl))tris(1H-pyrrole-2,5-dione; Figure 2b) was synthesised as described by Hanlon et al ((2017) Polymer Chemistry 8, 5120-5128) with minor modifications. A solution of maleic anhydride (0.59 g, 6.00 mmol) in anhydrous dimethyl formamide (DMF) (2.43 mL) was prepared under inert atmosphere and cooled to 0 °C. Separately, a solution of tris(2-aminoethyl)amine (0.29 mL) in dry DMF (2.04 mL) was prepared under inert atmosphere, and added dropwise to the maleic anhydride solution at 0 ^C over 30 minutes. The solution was stirred for a further 30 minutes at 0 ^C. A solution of sodium acetate (0.048 g, 0.60 mmol) in acetic anhydride (0.60 mL) was added to the reaction mixture at room temperature and stirred overnight at 50 ^C under inert atmosphere. The reaction mixture was concentrated using rotary evaporation, resuspended in DCM (50.00 mL) and washed with saturated brine (6 x 50.00 mL). The organic layer was collected, concentrated using rotary evaporation, resuspended in DCM (50.00 mL) and further washed with saturated sodium bicarbonate solution (6 x 50.00 mL). The organic layer was collected and concentrated using rotary evaporation to obtain the crude product. The crude product was purified using silica chromatography, 85:15 (ethyl acetate:hexane). The resulting pure yellow crystalline product was dried under vacuum overnight with a yield of 9% (0.21 g, 0.54 mM).¹H NMR (400 MHz, 298 K, DMSO-d₆): δ: 6.98 (s, 6H), 3.38 (t, J = 6.60 Hz, 6H), 2.60 (t, J = 6.62 Hz, 6H). NMR consistent with previously reported values (see Figure 4). Crosslinker compound NMR characterisation: NMR spectra for Compounds 1 and 2 were obtained on a Bruker AV2400 MHz spectrometer. The data was processed using TopSpin software. NMR chemical shift values are reported in parts per million (ppm) and calibrated to the centre of the residual solvent peak set (s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet). Electrospray ionisation mass spectrometry (ESI-MS) was performed on an Agilent HPLC system connected to a Bruker micrOTOF-Q mass spectrum instrument. Spectra were analysed using Bruker’s Compass Data Analysis software. All samples were run using solvent A (0.05% TFA in water) and solvent B (80% acetonitrile, 0.045% TFA in water). Samples were prepared at a concentration of 100 ^M peptide in phosphate buffer (20 mM NaH₂PO₄.2H₂O, 50 mM NaCl, pH 7.4) and reduced with 5 mM TCEP. Following a 10 minute reduction time, the respective compound was added at a 10:1 ratio and allowed to react for two hours. A total of 5 ^L of the sample was then loaded into a liquid chromatography mass spectrometry (LCMS) system. Figure 5 shows the LCMS characterisation of compounds 1 and 2, in which it was confirmed that all maleimide groups in both compound 1 and compound 2 were capable of binding biological macromolecules. pGEL Sequences: Bold = adaptions. Italic = expression tagged removed in production. C = cysteine attachment sites for attachment of the crosslinker. pGEL01 – engineered expression sequence of R1-R2-R3 rod domains: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSRGHMPPL TSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHSQVD AITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQ AAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGESDTDPHFQDVLMQ LANAVASAAAALVLKAKSVAQRTEDSGLQTQVIAAATQSALSTSQLVASTKVVAPTISSPVS QEQLVEAGRLVAKAVEGSVSASQAATEDGQLLRGVGAAATAVTQALNELLQHVKAHATGA GPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSR KLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGTGG GGSGGGGSGGGGSC (SEQ ID NO:16) pGEL02 sequence – engineered expression sequence of three copies of the R9 rod domain in series. MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAPGQLES ETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEA SQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGG NPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGGGGSAPGQLESETAIA ALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEASQLG HKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGGNPKQ AAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGGGGSAPGQLESETAIAALNS SLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEASQLGHKVS QMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGGNPKQAAHT QEALEEAVQMMTEAVEDLTTTLNEAASAAGGTGGGGSGGGGSGGGGSC (SEQ ID NO:17). pGEL03 sequence – engineered expression sequence of three copies of the R3 rod domain in series MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAHATGAG PAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRK LLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGGGG SGGGGSAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIK ADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMAT NAAAQNAIKKGGGGSGGGGSAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQ ARILAQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQ QRLREAAEGLRMATNAAAQNAIKKGTGGGGSGGGGSGGGGSC (SEQ ID NO:18) pGEL04 sequence – engineered expression sequence of three copies of the R1 rod domain in series MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSRGHMPPL TSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHSQVD AITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQ AAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGGGGSGGGGSRGHM PPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHS QVDAITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGR PLLQAAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGGGGSGGGGS RGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASKAWRKNKMDESK HEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGSAVTTISSNLTEMSRGVKLLAALLEDEG GNGRPLLQAAKGLAGAVSELLRSAQPASAEPRQNLLQAAGNVGQASGELLQQIGTGGGG SGGGGSGGGGSC (SEQ ID NO:19). pGEL05 sequence – engineered expression sequence of R9-R3-R9 rod domains MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAPGQLES ETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEA SQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGG NPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGGGGSAHATGAGPAG RYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRKLLS AAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGGGGSGG GGSAPGQLESETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLI EPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQ LLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGTGGGGSGGGG SGGGGSC (SEQ ID NO:20). pGEL06 sequence – engineered expression sequence of R9-R3-R9 rod domains MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSAHATGAG PAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRK LLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGGGG SGGGGSAPGQLESETAIAALNSSLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEI SHLIEPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAE SALQLLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGGGGGSGG GGSAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADA EGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNAA AQNAIKKGTGGGGSGGGGSGGGGSC (SEQ ID NO:21). pGEL07 sequence – engineered expression sequence of three copies of the engineered R13 rod domain in series MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGSGSDPTVIAE NELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQILEAAKSIAAATSALVKAASAAQRE LVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNNLSEAANAAVQGHASQEKLISSAK QVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAAAFEDGGGGSG GGGSDPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQILEAAKSIAAATSA LVKAASAAQRELVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNNLSEAANAAVQGH ASQEKLISSAKQVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAA AFEDGGGGGSGGGGSDPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQI LEAAKSIAAATSALVKAASAAQRELVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNN LSEAANAAVQGHASQEKLISSAKQVAASTAQLLVASKVKADQDSEAMKRLQAAGNAVKRA SDNLVKAAQKAAAFEDGGGGSGGGGSGGGGSC (SEQ ID NO:22). N-terminal adaptation – CGGGGSGGGGSGGGGSGS (SEQ ID NO:10) C-terminal adaptation – GGGGSGGGGSGGGGSC (SEQ ID NO:12) Linker between non-sequential domains – GGGGSGGGGS (SEQ ID NO:8) Alignment of Talin Rod domains R1-R3 with the engineered pGEL version of R1-R3 (with the 5 cysteine residues engineered to serine): The sequence GIDPFT in the expression sequence is a cloning artifact from Tobacco Etch Virus (TEV) protease and the expression system used to produce the sequence. C = cysteine attachment sites for attachment of the crosslinker. S = cys/ser substitution. R1-R3 -------------------------------------------------- PGEL01 MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTGCGGGGSGGGGSGGGGS R1-R3 --RGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASK PGEL01 GSRGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFETLPPLGQDAASK ************************************************ R1-R3 AWRKNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGCAVTT PGEL01 AWRKNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGSAVTT *********************************************.**** R1-R3 ISSNLTEMSRGVKLLAALLEDEGGNGRPLLQAAKGLAGAVSELLRSAQPA PGEL01 ISSNLTEMSRGVKLLAALLEDEGGNGRPLLQAAKGLAGAVSELLRSAQPA ************************************************** R1-R3 SAEPRQNLLQAAGNVGQASGELLQQIGESDTDPHFQDVLMQLANAVASAA PGEL01 SAEPRQNLLQAAGNVGQASGELLQQIGESDTDPHFQDVLMQLANAVASAA ************************************************** R1-R3 AALVLKAKSVAQRTEDSGLQTQVIAAATQCALSTSQLVACTKVVAPTISS PGEL01 AALVLKAKSVAQRTEDSGLQTQVIAAATQSALSTSQLVASTKVVAPTISS *****************************.*********.********** R1-R3 PVCQEQLVEAGRLVAKAVEGCVSASQAATEDGQLLRGVGAAATAVTQALN PGEL01 PVSQEQLVEAGRLVAKAVEGSVSASQAATEDGQLLRGVGAAATAVTQALN **.*****************.***************************** ELLQHVKAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARIL PGEL01 ELLQHVKAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARIL ************************************************** R1-R3 AQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAA PGEL01 AQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAA ************************************************** R1-R3 HPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGT---------------- PGEL01 HPDSEEQQQRLREAAEGLRMATNAAAQNAIKKGTGGGGSGGGGSGGGGSC ********************************** Sequence for wild-type R1 derived from mouse talin 1 and including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTRGHMPPLTSAQQALTGTINSSMQAVQAA QATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAETDY TAVGCAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQAAKGLAGAVSELLRSAQPASA EPRQNLLQAAGNVGQASGELLQQI (SEQ ID NO:23) Sequence for mutated R1 derived from mouse talin 1 and including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTRGHMPPLTSAQQALTGTINSSMQAVQAA QATLDDFETLPPLGQDAASKAWRKNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAETDY TAVGSAVTTISSNLTEMSRGVKLLAALLEDEGGNGRPLLQAAKGLAGAVSELLRSAQPASA EPRQNLLQAAGNVGQASGELLQQI (SEQ ID NO:24). Sequence for wild-type R2 derived from mouse talin 1 and including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTIGESDTDPHFQDVLMQLAKAVASAAAALV LKAKSVAQRTEDSGLQTQVIAAATQCALSTSQLVACTKVVAPTISSPVCQEQLVEAGRLVAK AVEGCVSASQAATEDGQLLRGVGAAATAVTQALNELLQHVK (SEQ ID NO:25). Sequence for mutated R2 derived from mouse talin 1 and including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTIGESDTDPHFQDVLMQLAKAVASAAAALV LKAKSVAQRTEDSGLQTQVIAAATQSALSTSQLVASTKVVAPTISSPVSQEQLVEAGRLVAK AVEGSVSASQAATEDGQLLRGVGAAATAVTQALNELLQHVK (SEQ ID NO:26). Sequence for wild-type R3 derived from mouse talin 1 and including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTAHATGAGPAGRYDQATDTILTVTENIFSS MGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKG AAAHPDSEEQQQRLREAAEGLRMATNAAAQ NAIKK (SEQ ID NO:27). Sequence of green fluorescent protein including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMVSKGEELFTGVVPILVELDGDVNGHKF SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSA MPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDP NEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:28). Sequence of GFP-labelled vinculin VD1 including an expression sequence: MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMVSKGEELFTGVVPILVELDGDVNGHKF SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSA MPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDP NEKRDHMVLLEFVTAAGITLGMDELYKSGGSGPVFHTRTIESILEPVAQQISHLVIMHEEGEV DGKAIPDLTAPVAAVQAAVSNLVRVGKETVQTTEDQILKRDMPPAFIKVENACTKLVQAAQ MLQSDPYSVPARDYLIDGSRGILSGTSDLLLTFDEAEVRKIIRVCKGILEYLTVAEVVETMEDL VTYTKNLGPGMTKMAKMIDERQQELTHQEHRVMLVNSMNTVKELLPVLISAMKIFVTTKNS KNQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWDEDAW (SEQ ID NO:29). Protein engineering: The genes encoding pGEL01 (SEQ ID NO:16), pGEL02 (SEQ ID NO:17), pGEL03 (SEQ ID NO:18), pGEL04 (SEQ ID NO:19), pGEL05 (SEQ ID NO:20), pGEL06 (SEQ ID NO:21), pGEL07 (SEQ ID NO:22), wildtype R1 (SEQ ID NO:23), mutated_R1 (SEQ ID NO:24), wildtype R2 (SEQ ID NO:25), mutated_R2 (SEQ ID NO:26), wildtype R3 (SEQ ID NO:27), GFP (green fluorescent protein; SEQ ID NO:28) and GFP-labelled vinculin VD1 (GFP-VD1; SEQ ID NO:29) and were constructed in pET-151 vectors (GeneArt, ThermoFisher). The proteins were expressed in BL21(DE3)* E. coli (Fisher). Protein purification was achieved using HisTrap HP columns (Cytiva®) for His-tag based affinity chromatography using an AKTA start protein purification system (Cytiva). Following purification, proteins were dialysed in phosphate buffer (20 mM NaH2PO4.2H2O, 50 mM NaCl, pH 7.4). Protein expression and purification: pGEL01, GFP-VD1, wildtype R1, mutated_R1, wildtype R2, mutated_R2, wildtype R3 and GFP were transformed into BL21(DE3) cells. Overnight cultures were used to inoculate Luria-Bertani Broth (pH 7.2) or M9 minimal media with ¹⁵N ammonium chloride for labelled samples, which was subsequently grown at 37 ^C until an OD₆₀₀0.6-0.8 was reached. The culture was then induced with 100 ^M isopropyl β- d-1-thiogalactopyranoside (IPTG) and grown overnight at 20 ^C. Harvested cells were resuspended in nickel buffer A (20 mM Tris, 500 mM sodium chloride, 20 mM imidazole), sonicated and centrifuged (6,000 RPM, 40 minutes, 4 ^C). The supernatant was then loaded onto a HP HisTrap Nickel column (Cytiva®) connected to an AKTA start system (Cytiva®) and eluted with nickel buffer B (20 mM Tris, 500 mM sodium chloride, 500 mM imidazole). Resulting pure protein was dialysed (10 kDa MWCO) into phosphate buffer (pH 7.4) overnight, ready for use. Samples were cleaved with TEV protease following HisTrap Nickel column and dialysed into Q buffer A (20 mM Tris, 50 mM sodium chloride). The resulting dialysed sample was loaded onto a HiTrap Q HP column connected to an AKTA start system (Cytiva®) and eluted with Q buffer B (20 mM Tris, 1M sodium chloride). Resulting pure protein was dialysed into phosphate buffer (20 mM NaH₂PO₄.2H₂O, 50 mM NaCl, pH 7.4) overnight, ready for use. In a separate experiment, the pGEL01, pGEL02, pGEL03, pGEL04, pGEL05 and pGEL06 (see Table 1 below) were also expressed in minimal media containing ¹⁵N-labelled ammonium chloride to produce ¹⁵N-labelled proteins and purified by Ni affinity and anion exchange (Fig.6A-F). All monomers were expressed in minimal media and purified well. Some contaminants were seen even after anion exchange purification, but the amount was relatively small compared to the monomer bands and not enough to affect NMR and circular dichroism experiments. Table 1: summary of monomers and rod domains in each monomer. The unfolded forces were measured as per Yao, M. et al.2016 (supra). M p p p p p p p In another experiment, expression was first performed under the above conditions, using complex Luria-Bertani Broth medium at expressing at 18°C following induction of gene expression (Figure 7A). An additional growth medium, Terrific Broth, which is rich and complex in nature, was used to culture the cells at either 37°C (Figure 7B) or 25°C (Figure 7C). Terrific broth was chosen as it is similar to the rich medium used in batch type fermentation experiments. Under the standard conditions where expression was performed in Luria-Bertani Broth medium at 18°C, pGEL01 was highly expressed following 21 hours post-induction (Figure 7A). Expression in Terrific Broth showed lower protein yield after 21 hours post-induction than Luria-Bertani Broth in both experiments. Nevertheless, culturing in Terrific Broth at 25°C was able to achieve similar expression to Luria-Bertani Broth at 2 hours post-induction (Figure 7C). Expression in Terrific Broth at 37°C was able to achieve comparable or higher protein yield at 2- and 4-hours post-induction to Luria-Bertani Broth (Figure 7B). The next experiment investigated the use of a difference crosslinking mechanism, using the Spy technology. According to Wikipedia, the SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair. DNA sequence encoding either SpyTag or SpyCatcher can be recombinantly introduced into the DNA sequence encoding a protein or amino acid sequence of interest, forming a fusion protein. These fusion proteins can be covalently linked when mixed in a reaction through the SpyTag/SpyCatcher system. In this way, bioconjugation can be achieved between two recombinant proteins that would otherwise be restrictive or impossible with traditional direct genetic fusion between the two proteins. To explore this technology, a pair of monomers were designed to be used together, one containing pGEL01 gene with two SpyTag peptides at each terminus separated by flexible linkers, and another containing pGEL01 gene with a SpyCatcher peptide at each termini (Figure 8). By addition of four SpyTags per monomer (Figure 8B), one SpyTag monomer would have the capability to bind multiple SpyCatcher monomers, in theory creating crosslinking upon mixing (Figure 8C). Expression of the pGEL01 monomers with Spy peptides was tested in 750 mL LB media then cells harvested and purified by nickel affinity following overnight expression (Figure 9). The SpyCatcher-pGEL01 monomer showed a good protein yield, such that it exceeded the nickel column capacity, and some protein was lost in the flow through (Figure 9A). However, the SpyTag-pGEL01 showed a poorer protein yield under the conditions used (Figure 9B). Circular dichroism (CD) and ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) were used to confirm that the R1-R3 domains of talin incorporated in pGEL01 retained the alpha helical folding of the wild type talin domains. All circular dichroism experiments were performed on the JASCO J-175 spectropolarimeter using a 1 mm pathlength quartz cuvette. Far UV-spectra were obtained between 200-260 nm with an average of 4 scans at 100 nm/min, 0.5 nm step resolution, 1.0 second response and 0.5 nm bandwidth. For spectral scans CD bandwidth was set to 222 nm. Measurements were taken between 20-90 ^C with 20 second step resolution, 4 seconds of response and 1.0 nm bandwidth. Samples were prepared between 20-50 ^M in 400 ^L of phosphate buffer (20 mM NaH₂PO₄.2H₂O, 50 mM NaCl, pH 7.4). Protein NMR experiments were conducted at 298 K using a Bruker AVANCE III spectrometer equipped with a QCI-P cryoprobe.¹⁵N-labelled proteins were measured at 150 ^M in 20 mM phosphate, 50 mM sodium chloride, 2 mM DTT and 5% v/v ²H₂O at pH 6.5. Spectra were processed with TopSpin and CcpNmr Analysis 2.5.2. As shown in Figure 10, circular dichroism confirmed preserved alpha helical folding between pGEL01 and wild type R1-R3 domains of talin. Figure 11a shows that alpha helical folding was also retained in the mutated R2 following four cysteines mutated to serines compared to wild type R2. As seen in Figure 11b, a change in T_m was seen between the mutated R2 and wild type R2, suggesting minor alterations to stability had occurred. The NMR spectrum of pGEL01 showed many peaks (Figure 12A), unsurprising due to the presence of three different rod domains. There was clear overlay with the spectrum of ¹⁵N- labelled mouse Talin1 R3 at certain peaks indicative of R3 domain (Figure 12B). The NMR spectrum of pGEL02 showed sharp peaks (Figure 13A), which overlaid well with a spectrum of ¹⁵N-labelled mouse Talin1 R9 (Figure 13B). NMR spectrum of pGEL03 also has sharp clear peaks (Figure 13C) and overlaid well with mouse talin1 R3 spectrum (Figure 13D). The NMR spectrum of pGEL04 showed sharp, clear peaks (Figure 14A) which overlaid well with the spectrum of ¹⁵N-labelled mouse Talin1 R1 (Figure 14B). pGEL05 and pGEL06 were designed to investigate properties of engineered monomer containing different rod domains, specifically R3 and R9 domains. NMR spectra for both pGEL05 (R3-R9-R3) and pGEL06 (R9-R3-R9) have sharp signals but the pGEL06 spectrum had significantly fewer peaks than pGEL05, although both pGEL05 and pGEL06 were expected to have similar peak patterns as they are both comprised of R3 and R9 domains (Figures 15A and B). The spectrum of pGEL05 overlay well with both R3 and R9 spectra (Figures 15C and D), but pGEL06 overlays well with the R3 spectrum, but unexpectedly the peaks from R9 are not clearly evident indicating that the folding of R9 has been impacted in this pGEL monomer (Figure 15E). Circular dichroism (CD) datasets were also collected for pGEL02, pGEL03, pGEL05, and pGEL06 to investigate their folding changes and thermal stabilities. These engineered monomers have alpha helical folding at 20°C, and the structures were completely lost at 90°C (pGEL02 and pGEL03) or 95°C (pGEL05 and pGEL06) (Figures 16A-D, lower panels). pGEL02 presented a T_m of 51.0°C (Figure 16A, upper panel) and pGEL03 showed much higher stability at high temperature (Figure 16B, upper panel), which is to be expected as the R3 domain is relatively thermally stable. The T_m of pGEL03 was 76.2°C. Melting curves of pGEL05 and pGEL06 showed two melting steps which were caused by the two different rod domains in them unfolding separately (Figures 16C and D, upper panels). The first melting phases were presenting R9 domain(s) in the monomers and the second phases were responsible for R3 domain/domains unfolding steps. Interestingly, these two novel monomers showed different thermal stabilities even though they were made with the same rod domains, comparing pGEL05 with Tm1 of 47.6°C and Tm2 of 81.3°C and pGEL06 with Tm1 of 41.7°C and Tm2 of 76.8°C. CD datasets were also collected for pGEL01 and pGEL04 to investigate their folding changes and thermal stabilities. The melting curve of pGEL01 showed three distinct melting steps (Figure 17A), with T_m1 occurring at 63.3°C, T_m2 at 68.5°C and T_m3 still not completely unfolded within the temperature range tested. CD scan data confirmed the helical structure, which was mostly gone at 95°C (Figure 17B). The melting curve for pGEL04 showed only one unfolding step occurring at 68.9°C (Figure 18A), as expected due to the presence of only one type of rod domain. CD scan data confirmed the helical structure, which was mostly gone at 95°C (Figure 18B). Figure 19 shows the melting curve of mouse talin1 R13 showing that R13 was still in the middle of unfolding phase at 90°C. R13 is known to have a similar mechanical strength to R9, while thermally stable rod domain R3 is known as the weakest rod domain under mechanical force. The monomer, pGEL07 having three R13 rod domains in series, was generated because thermal and mechanical stabilities are key functions of the monomers described herein and their ability to make suitable hydrogel polymers. 2D HSQC spectra of ¹⁵N-labelled mutated R1 (Figure 20) show that folding was retained compared to the wild type R1 from Banno et al (Journal of Biological Chemistry (2012) 287, 13799-13812) overlaid and that very little change to the structure occurred from the single mutation in the mutated R1. Figure 21 is the HSQC spectra of ¹⁵N-labelled mutated R1 with wild type R1 assignments from Banno et al (supra) overlaid, centred on the region where the cysteine in wild type R1 was positioned. Effects of protein concentration on resulting Talin Shock Absorbing Material (TSAM): In this experiment, a monomer (pGEL01) concentration of 300 mg/mL was tested in comparison with 200 mg/mL used in previous experiments. Rheometry was performed at a temperature of 25°C on a single piece of TSAM. Five amplitude sweeps were performed in total and the data from each amplitude sweep are shown in Figure 22. From the rheology data collected for the 300 mg/mL TSAM at 25°C, the loss modulus (G”, red) was seen to begin at around 10 Pa at the start of the first sweep (Figure 22A) but increases to around 300 Pa by the start of the second sweep (Figure 22B), continuing to increase to over 1000 Pa by the start of the third sweep (Figure 22C) and reaching a maximum of 10,000 Pa in the fifth sweep (Figure 22E), mimicking the pattern shown by 200 mg/mL TSAM. Thus, this indicates that the TSAM material retains its properties upon increasing concentration. Unlike the results of the temperature ramp experiment at 200 mg/mL there is no gel-sol transition recorded under these experimental conditions and initial analysis would suggest that the shock absorbing properties should be retained at this higher concentration. Using compound 1 as a control and compound 2 as a crosslinking agent (Figures 2 and 23a), pGEL01 (Figure 23b) was formed into a hydrogel (Figures 23c-d) via tri-substitution of terminal cysteines with compound 2 (see Figure 5). It was hypothesised that, upon application of force (i.e. shear strain or impact), the three rod domains within each protein monomer (talin domains R1, R2, R3 with internal cysteines mutated to serines) would unfold, dissipating energy through the endothermic process of protein unfolding (Schon, A. et al (2017) Proteins-Structure Function and Bioinformatics 85, 2009-2016; (Figures 23e-h). The R1-R3 domains of talin in pGEL01 provide a stepwise unfolding when exposed to force, with the wild type domains exhibiting threshold unfolding forces of 20, 15 and 5 pN respectively (Yao, M. et al supra). The resulting talin shock absorbing material (TSAM) is a hydrogel that contains monomeric units capable of refolding upon removal of force, retaining its energy dissipating mechanism following any potential impact events. Due to the endothermic energy dissipating mechanisms in the TSAM, the heating of the captured projectiles seen in aerogels would not be observed, offering a solution to several of the limitations seen with current state of the art impact absorption materials. Specifically, a recombinant form of the mechanosensitive protein talin was incorporated into a monomeric unit and crosslinked, resulting in the production of a TSAM. When subjected to 1.5 km/s supersonic shots, TSAMs were shown not only to absorb the impact, but to capture/preserve the projectile. Engineered Talin Shock Absorbing Material (TSAM) structural characterisation Following formation of a TSAM using pGEL01 monomers, characterisation of the internal network structure was conducted. Preparation: A 30:1 ratio of TCEP:cysteine was slowly added to a solution of pGEL01 in phosphate buffer (pH 7.4). After one hour, the pGEL01 solution was run through PD10 desalting columns (Cytiva) and the flow through was then re-applied to desalting columns for a second run through to ensure TCEP removal. Immediately following the desalting step, the pGEL01 solution was concentrated to the desired concentration using 30 kDa MWCO concentrators (Sigma). The TSAM was then formed through the addition of compound 2 at 1:1 maleimide:sulfhydryl. Samples were left to set at 4 ^C overnight. Immuno-gold staining and transmission electron microscopy: A 2 µl of sample was applied to carbon/formvar 400 mesh gold grids (Agar Scientific) and allowed to settle on the grid for 5 minutes. The sample was then fixed in 2% formaldehyde and 0.5% glutaraldehyde in 100 mM sodium cacodylate buffer (CAB) pH 7.2 for 15 minutes at room temperature. Samples were washed 2 x 5 minutes in CAB and 2 x 5 minutes in 20 mM Tris, 500 mM NaCl, 0.1% BSA and 0.5% Tween 20 (TBST). Grids were blocked in 2% BSA in TBST for 30 minutes and then moved into a 20 µL drop of anti His-Tag primary antibody (Sigma) diluted 1:100. Grids were washed 6 x 2 minutes in drops of TBST before incubation in Goat anti-mouse IgG conjugated to 5 nm gold (British Biocell International) diluted 1:50 for 30 minutes. Grids were washed for 6 x 2 minutes in TBST and 6 x 2 minutes in distilled water. Negative controls were performed as above but primary antibody was replaced with TBST. Samples were then air dried and negative stained in 2% aqueous uranyl acetate. Samples were viewed in a Jeol 1230 Transmission electron microscope at 80 kV and images were recorded on a Gatan One View 16 MP digital camera. Scanning electron microscopy: The TSAM was dehydrated to form a xerogel and placed on a carbon tab mounted onto an aluminium stub. Imaging was achieved using a Hitachi S-3400N scanning electron microscope with elemental dispersive X-ray analysis and analysed using Oxford instruments AZtec software. His-tagged gold immunostaining of the TSAM, imaged using transmission electron microscopy (TEM), confirmed the presence of pGEL01 in a lattice formation, displaying pore sizes of approximately 100 nm (Figure 24a). Following this, scanning electron microscopy (SEM) revealed the TSAM to contain a porous like structure on the micrometre scale typical of hydrogels (Figure 24b), with long fibres of width ≈2 µm and pores of ≈10 µm. Elemental dispersive X-ray (EDX) analysis confirmed the observed fibres in the SEM images consisted of sulphur and carbon (Figure 24c), pGEL01 representing the only component of the xerogel containing these atoms. Together these findings indicated pGEL01 molecules linked with compound 2 form a lattice on the nanometre scale, morphing into larger fibrillar like structures on the micrometre scale. When handling the TSAMs, high levels of extensibility were observed, presenting extension of >3-fold when under tension, and returning to original size upon removal of force (Figure 124d-e). Evidence for pGEL01 domain unfolding in TSAM Rheological measurements: Rheological measurements were performed on an Anton Parr modular compact rheometer (MCR302). All measurements were performed at 298 K using a PP20 parallel plate. Oscillatory amplitude experiments maintained a frequency of 10 rad s^-1 and were performed with an amplitude of oscillation range of 0.01% to 100%. A 2 minute rest time was set between each amplitude sweep, with a total of five sweeps performed on each TSAM. For the GFP-VD1, GFP and buffer swelled experiments, the TSAM was left in the respective solution at 2 mg/mL overnight before rheological measurements were performed. Subsequent rheological characterisation of TSAMs provided strong evidence for the induced unfolding of the talin domains within the material when exposed to shear strain, indicating that the energy dissipating mechanisms of talin were successfully incorporated into TSAMs. Specifically, for the first applied oscillatory shear strain sweep on the TSAM, the dynamic shear storage (G’) and loss modulus (G”), as a product of shear strain presented a linear viscoelastic region (LVER) extending across the full range of shear strain tested (Figure 24f). Following a two-minute recovery period, a subsequent sweep was performed, once again followed by a two-minute rest period. This protocol was conducted for a total of five sweeps. Owing to the unfolding and refolding kinetics intrinsic to R1-R3 (pGEL01), it was predicted that viscoelastic properties would be retained upon repeated exposure to shear strain. The TSAMs presented G’ > G” for all five sweeps, indicating viscoelastic behaviour during the entirety of the five consecutive oscillatory shear strain sweeps. Moreover, the complex modulus (G*) (sum of G’ and G”) increased concomitantly with accumulated sweeps (Figure 24f), indicating the TSAM presented increased resistance to deformation upon repeated exposure to shear strain (Kulkarni, V.S. & Shaw, C. in Essential Chemistry for Formulators of Semisolid and Liquid Dosages. (eds. V.S. Kulkarni & C. Shaw) 145-182 (Academic Press, Boston; 2016)). Strain stiffening as a consequence of fibre reorganisation is a well-documented phenomenon occurring in hydrogels formed from biopolymers (Wen, Q. & Janmey, P.A. (2011) Current Opinion in Solid State & Materials Science 15, 177-182), causing the elastic modulus to increase with strain. Strain stiffening presents here as the positive gradient observed in sweeps 3-5, whereby a peak is reached between 0.1-10.0% strain (Figure 24f). Specifically, the increase in G*, presented as a bell-curve for both G’ to G” on sweeps 3-5, with the peak occurring between 0.1-10% shear strain (Figure 24f). For clarity, only G’ is discussed herein. Graphs also containing G” are found in Figures 25a-e. The peak maxima for G’ was reached between 1-5% for sweeps 2-5, shifting to the right and increasing in amplitude for each subsequent sweep. Without wishing to be bound by theory, it is proposed that this phenomenon is due to strain stiffening, resulting in a tighter, more rigid network structure with more of the talin domains arranged in parallel to the axis of the fibres (Figure 24i). As a consequence of the increased network rigidity, strain can become imparted on the internal structure of the fibres themselves. When a maximum fibre strain is reached, mass chain unfolding of the protein domains occur, causing the positive slope to transition into a negative gradient as a result of the sudden introduction of slack from the extension of the proteins (Figure 23e-g and Figure 24i). Due to the sudden introduction of slack, a lag between the controlled shear strain and measured shear stress sine waves occurs, presenting as a sudden shift towards more viscous-like behaviour, hence the negative gradient of G’ and decrease in G* (Figure 24f). Alongside the observed bell-shaped trend of G’ and G” with accumulated sweeps, an increase in phase angle was observed, also presenting a bell-curve (Figure 24g). Interestingly this pattern was shifted to higher strains, with the peak of the bell occurring between 10-15% strain, directly correlating with the negative slope of G’. The large quantity of slack (Figure 24i) introduced into the system following a mass unfolding event allows for increased flow, observed as a drastic phase shift towards 45 degrees, and decreased rigidity, seen as a drop in G*. With increased application of shear strain, this phase shift peak eventually declines as tension within the fibres begin to increase again. Upon removal of shear strain, unfolded proteins may then refold, and the resulting TSAM displays an enhanced rigidity (higher G* at the start of the subsequent sweep) due to the reorganised network of fibres. Shear stress vs. shear strain correlations corroborate these results, revealing an exponential increase in shear modulus (G), a measure of rigidity, with accumulated sweeps, further illustrating the strain stiffening within TSAM (Figure 24h). Furthermore, sweeps 4 and 5 reached apparent shear yield points, beginning to move into viscous stress as seen by the induction of a slope, subsequently transitioning back into a linear gradient indicating the reoccurrence of elastic behaviour. These results summarise the previously described phenomenon in a single graph (Figure 24h), with linear elastic regions indicating the reordering of the network structure and gradual tension occurring on the fibres, the slope indicating sudden mass unfolding of talin rod domains, and subsequent linear elasticity reoccurring with tension once again building on the fibres with the increased shear strain. To confirm that the unfolding of the talin domains within the TSAM was directly responsible for the rheological characteristics/material properties observed, a green fluorescent protein tagged-vinculin domain 1 protein (GFP-VD1) was employed. The effects of temperature on a single piece of an engineered TSAM using 200 mg/mL pGEL01 were investigated using a rheometry temperature ramp. Five amplitude sweeps were performed per temperature over a range between 10°C and 85°C. The data from first and last amplitude sweeps for each temperature are shown in Figure 26. After setting the TSAM overnight at 10°C, the first five sweeps were performed at the same temperature. The storage modulus, G’, was measured along with the loss modulus, G”. Where G’ was greater than G”, the material was confirmed to be a gel. Where G” was greater than G’ the material under analysis was deemed to have undergone a gel-sol transition and now to adopt solution state properties. In the 10°C amplitude sweeps, G’ remained above G” loss modulus for the first and last sweeps, indicating the TSAM began and ended as a gel. However, these values became closer towards 100% shear strain and there is evidence that the material may have been approaching a gel-sol transition. But, the properties of the material returned before the next amplitude sweep was performed, with an increase in G’ and decrease in G’’ observed as the consecutive sweeps were performed (Figures 26A-B). At 25°C, a gel-sol transition was observed after five sweeps had been performed, where the storage modulus (G’, blue) dropped rapidly after 10% shear strain (Figure 26D). It is also worth noting that, at 25°C, there was an observable increase in the values of both moduli during the progression from sweep 1 (began at around 700 Pa, Figure 26C) to sweep 5 (began at around 2000 Pa, Figure 20D). However, the gel material properties were returned to the material before subsequent analysis was performed at 35°C. In the 35°C amplitude sweeps, the TSAM began again as a gel (Figure 26E), indicating the structure had not been destroyed despite performing as a solution at the end of the last sweep at 25°C. By the fifth 35°C sweep, the material no longer appeared to demonstrate any response to increasing shear strain (Figure 26F). At 45°C, the TSAM also demonstrated little change in material properties upon the application of repetitive increases in shear strain up to 10% (Figures 26G-H). After this point, G’ and G’’ began to decrease proportionally to one another, with material properties returning before the next consecutive sweep commenced. At this point, visual inspection of the TSAM revealed the material hardening. By the 65°C sweeps, there was no indication of TSAM melting as expected, according to pGEL01’s melting profile as characterised by circular dichroism (Figures 26I-J). Following the 85°C sweeps, the TSAM had completely hardened, but the material properties were retained as they were at 45°C. For the next stage of the experiment, a temperature ramp was performed in a plate reader to acquire simulation state data on the structure of fibres. The plate reader was capable of heating between 25°C and 45°C. An optical density (OD) scan to measure at wavelengths between 300 – 1000 nm was performed on triplicate samples of pGEL01 before adding a tripodal maleimide crosslinker (Formula I) then, after setting, heating to 25°C, then 35°C then 45°C with wavelength scans repeated at each temperature. In particular, three repeats of 100 μL pGEL01 were placed in a 96 well plate along with controls of PBS buffer. An optical density scan was performed at wavelengths between 300 – 1000 nm in a plate reader at 25°C before the addition of crosslinker, using PBS as a blank. After setting overnight, the resulting TSAM was heated sequentially in the plate reader to 25, 35 then 45°C and optical density measured at each temperature. Optical density data was plotted against wavelength for each condition and is shown in Figure 27. The data showed pGEL01 pre- and post-crosslinker addition absorbs highly around 300 nm. Although maleimides, like the crosslinker, reportedly absorb highly around 300 nm, the data shows pGEL01 before crosslinker addition also absorbs highly at this wavelength. This could be due to absorbance of certain amino acids around 280 nm. A small peak is observed around 410 nm, and another small peak at 660 nm, but neither appear unique to either crosslinked or uncrosslinked pGEL01. Statistical analysis has not yet been performed. Samples from a sacrificial well in the plate reader were obtained after each temperature ramp and taken forward for Fourier-transform infrared (FTIR) spectroscopy along with the remainder of the sample from the rheometer which was desiccated from heating to 85°C. Data was normalised against a blank of PBS buffer, then deconvoluted using OriginPro software. To perform the peak deconvolution, peak locations were manually picked in OriginPro software and chosen based on their ability to give the best cumulative peak fit line to the absorbance datapoints in the lowest number of peaks. Therefore, for some datasets, two peaks were chosen and for others three peaks were chosen. The software then calculated the peak maxima of each predicted peak. Deconvoluted peak data is show in Figure 28. FTIR analysis of all heated samples showed a similar pattern of peaks; all showed a predominant peak between 1640 - 1644 cm^-1, corresponding to the presence of unordered structures (Figures 28A-E). This is likely due to the crosslinked, overlapping strands present in the gel matrix. All samples also showed a peak between 1645 – 1650 cm^-1, corresponding to the presence of α-helical structures, contained in the engineered talin protein monomers and previously confirmed by NMR (Figures 28A-E). Some samples showed small peaks picked at 1683 cm^-1, corresponding to aggregated strands (Figure 28C red peak; Figure 28D blue peak). However, plots from other samples also show a small peak in a similar region even if not picked for deconvolution (Figures 28A, B, E). This is likely to be a small amount of protein aggregation formed during TSAM preparation, due to the high concentration of protein. Rheometry data collected using a single sample of set TSAM showed slightly lower moduli when amplitude sweeps were performed at 10°C compared to higher temperatures. This observation provides some early indication of TSAM’s resistance to strain at lower temperatures. Previous data (Doolan J.A. et al (2023) Nat. Nanotech.18, 1060–1066) has indicated that TSAM’s resistance to strain improves with repeated strain, as the position of the gel fibres are optimised, rigidifying the material. Amplitude sweeps at 25°C and 35°C demonstrated TSAM’s rather unique gel-sol-gel transition characteristic, where it is able seemingly to reform to a gel after performing as a solution in response to high strain. As noted at 25°C, the increase in the values of both moduli during the progression from sweep 1 to sweep 5 do support other findings that TSAM’s shock absorbing properties may increase with repeated strain. Interestingly, FTIR data demonstrated remarkably similar structural profiles for all TSAM samples, even the sample that had been heated to 85°C and dried out. All samples retained alpha helical structures and there appeared to be no increase in unordered or aggregated structures as a result of heating or applying strain, in the case of the 85°C sample. Gel Filtration: Gel filtration was performed at room temperature using a Superdex 200 increase size- exclusion column (GE healthcare) system at a flow rate or 0.75 mL min^-1. Samples were run at 150 ^M, or at 450 ^M for the 3:1 condition, all at final volumes of 100 ^L in 20 mM tris, 150 mM sodium chloride, 2 mM DTT (pH 8.0). As illustrated in both Figure 29 and Figure 31a, GFP-VD1 is capable of selectively binding to the unfolded state of each of the rod domains, preventing domain refolding and ‘locking’ the extended domain confirmation in place (Yao, M.X. et al (2014) Scientific Reports 4, 4610). Here the GFP-VD1 was introduced into the pGEL01 TSAM pre-amplitude sweep as a 2 mg/mL solution through a material swelling process. The rheological properties of these materials were then elucidated and compared to the results of analogous studies in which the same TSAM material underwent the same material swelling process in a solution of GFP or buffer only. The resulting G’ and G” as a product of shear strain for the three conditions tested is summarised in Figures 30a-e. When plotted as shear stress against shear strain (Figure 31b) the GFP and buffer controls presented the same linear trend as attained in the first amplitude sweep of the non-treated TSAMs, indicating purely elastic behaviour. In contrast the TSAM treated with GFP-VD1 reached a yield point between 46-68% shear strain (Figure 31b) indicating the presence of protein unfolding events, re-entering the linear elastic region until it reached 100% strain. After sweep 1, all three conditions deviated from the non-treated TSAMs for sweeps 2-5, with no significant increase in G* observed between sweeps 1-5. This lack of increased G* between sweeps is attributed to the increased hydration of the TSAMs in the swelled state, reducing the density of the protein fibres constituting the TSAM and consequently limiting reorganisation of the network fibres occurring due to the decreased opportunity for neighbouring fibres to interact. However, whilst both GFP and buffer treated TSAMs remained fully elastic across all five strains, the GFP-VD1 treated condition reached its yield stress (between 46-68% shear strain), indicating unfolded pGEL, in every consecutive sweep. From previous rheology sweeps this trend is believed to be indicative of unfolded protein. However, in this instance, no increase in G* was observed beforehand, suggesting unfolding was independent of tension, as expected with GFP-VD1 binding limiting talin rod domain folding/unfolding. To confirm further the binding of GFP-VD1 to the TSAM fibres, a series of comparative fluorescence microscopy experiments were conducted. Fluorescence microscopy: GFP-VD1 and GFP treated samples of the TSAM made with pGEL01 monomers from the rheology experiments were visualised using an Olympus IX71 microscope employing a 1.6x magnification Optovar in combination with a PlanApo 100x OTIRFM-SP 1.49 NA lens mounted on a PIFOC z-axis focus drive (Physik Instrumente, Karlsruhe, Germany), and illuminated using LED light sources (Cairn Research Ltd, Faversham, UK) with DC/ET350/50x excitation, ET Quad Sedat dichroic, and DC/457/50m emission filters (Chroma, Bellows Falls, VT). Samples were visualised using a QuantEM (Photometrics) EMCCD camera, and the system was controlled with MetaMorph software (Molecular Devices). Each 3D-maximum projection of volume data was calculated from 31 z-plane images and the best 6 were chosen, each 0.2 µm apart, and analysed using MetaMorph software. Here, fibre like structures exhibiting the same diameter as those observed in the previous SEM studies (Figure 24b) were found to have localised GFP-VD1 (Figures 31c-d), confirming binding. In contrast, the GFP control treated TSAM fibres appeared as darker regions, with void spaces presenting higher GFP concentrations (Figures 31e-f), confirming an absence of GFP-VD1 binding events. TSAM captures and preserves projectiles from supersonic impacts Following the rheological evidence for TSAMs, retention of talin’s endothermic energy dissipation mechanism, the performance of the TSAM as an impact absorbing material was tested, probing TSAMs performance upon supersonic projectile impact. Light gas gun (LGG) experiments: The impact experiments were carried out using the Light Gas Gun facility at the University of Kent, Canterbury. The LGG is capable of accelerating projectiles smaller than 3.5 mm to speeds up to 7 km/s (Burchell, M.J. et al (1999) Measurement Science and Technology 10, 41-50.; Hibbert, R. et al (2017) 14th Hypervelocity Impact Symposium (Hvis 2017) 204, 208- 214). The TSAM target was set in a blast tank exit aperture (BTEA) with a circular, 8 mm diameter aperture, sealed with tape, with an aluminium (5083) back plate placed behind. Multiple 20-70 ^m basalt particles were loaded into a single sabot utilising the “buckshot” method and were fired at roughly 1.5km/s, with the speeds recorded via the BTEA - Muzzle laser method as described by Burchell et al (supra). The target was removed prior to the air flushing procedure to reduce gun contamination on the TSAM. The combination of the BTEA and target mount into a single device, allowed for minimal spreading of the buckshot projectile, increasing the chance of direct impact onto the TSAM, and maximized the BTEA- muzzle separation. Specifically, velocities of 1.5 km/s were tested, as this velocity has relevance in the aerospace industry and to dissipate ballistic/force impact (Williamsen, J. et al Vol.2109 6065, 1-10; Couldrick, C. (2012) in Advances in Military Textiles and Personal Equipment. (ed. E. Sparks) 196-212 (Woodhead Publishing)). For instance, particles in space impact both natural and man-made objects at speeds >1 km/s (Williamsen, J. et al supra), while muzzle velocities from firearms commonly fall between 0.4-1.0 km/s (Couldrick, C. supra). Here the TSAM, in addition to a commercially available polyvinylpyrrolidone hydrogel control, was placed in the target chamber of a light gas gun (LGG) and the following material properties investigated: (1) the ability of the TSAM to survive impact; (2) the ability of the TSAM to reduce the force of the projectile before impacting an aluminium back plate; and (3) the ability of the TSAM to capture the projectile in a preserved state. Spherical basalt particles between 20-70 µm were used as projectiles, loaded in a sabot as buckshot. A schematic for this experiment is given in Figures 32a-c. When shot at 1.5 km/s, the control gel was destroyed (Figure 32d), with a visible hole in the tape behind the gel (Figure 32e), and a crater of 1.33 mm in diameter produced in the aluminium back plate (Figure 32f). Therefore, this material control showed no detectable impact absorption properties. Instead, this control experiment demonstrates the effects of the kinetic energy contained in the projectiles. Under the same experimental conditions, the TSAM appeared mostly intact from the frontal perspective (Figure 32g), with no projectile permeation detected to either the supporting tape (Figure 32h) or the aluminium backplate (Figure 32i). In addition, subsequent SEM analysis identified the basalt particles embedded in the TSAM post shot (Figure 32j), confirming that the TSAM had completely absorbed the impact of the basalt buckshot. The transparency of the TSAM shown in Figure 32h and Figure 33 is an additional desirable property, allowing for the easy removal of caught projectiles from the TSAMs. To determine conclusively whether TSAM also enabled preservation of the captured basalt projectiles, SEM was performed on the impacted TSAM. Multiple basalt particles presenting a preserved circular shape were observed in the gel (Figures 32j-k), confirmed as basalt with EDX analysis (Figure 34). Thus, confirming TSAM also enables the preservation of the projectiles. Moreover, during one of the TSAM shots, shrapnel from the aluminium (Al 7075) burst disk (Figure 32b) appeared to strike the TSAM in combination with the basalt, as confirmed through SEM and EDX analysis (Figures 32l and 35). Such an impact often destroys the aerogel materials used within the aerospace industry for projectile capture, providing preliminary evidence that TSAMs are also able to overcome this industry standard material limitation. Research into the field of marketed armour demonstrated that some products use materials such as aramid fibres and ceramic layers to confer stab or blast protection. The next experiments investigated the effect of the addition of the TSAM of the present invention to such materials. A TSAM made using pGEL01 was applied to Twaron yarn (Teijin Aramid), supplied by National Institute of Standards and Technology, as well as to aluminium oxide ceramic beads. The aim of these experiments was to test the TSAM’s capability as an adhesive for these materials. As shown in Figure 36A, prior to addition of TSAM, the aramid fibres were easily separated into strands. TSAM was applied to the aramid fibres or ceramic beads immediately after addition of the crosslinker then left to set at 10°C overnight. The next day, the results were observed. The aramid fibres appeared bonded together by a film of TSAM, which had dried (Figure 36B). Upon manipulation, the aramid strands were firmly held together by TSAM. The ceramic beads were adhered together strongly by TSAM, and also adhered to the surface of the container (Figure 36C). Force applied to remove the ceramic beads from the container caused the surface of the ceramic to break. The beads could also be separated from each other by force, also breaking the surface of the ceramic (Figure 36D). The materials were then taken forward for imaging by scanning electron microscopy (SEM). SEM imaging of the aramid fibres adhered by TSAM revealed the fibres embedded within TSAM (Figure 37), where the TSAM shows some stretching/snapping behaviour. Elemental X-ray analysis showed the elemental distribution of the imaged material, where aramid fibres appear red indicating high carbon content, and the TSAM appears green/light blue indicating high sulphur and oxygen content (Figure 37B). SEM imaging of the ceramic bead surface showed an uneven surface indicative of breakage (Figure 38). Broken ceramic shards could be seen embedded within the TSAM material located on the breakage surface (Figure 38A). Lower magnification imaging in Figure 38B also showed a layer of ceramic (white) covering the layer of TSAM underneath (grey). These results confirm breakage of the ceramic rather than the TSAM as a result of applying force. Thus, TSAM demonstrates an unexpected phenomenon of forming a strong adhesive bond to aramid, ceramic and plastic materials. The film created by TSAM drying onto these materials was able to withstand forces in spite of no longer maintaining a gel consistency, conferring a glue-like property. Data obtained from ceramic bead adhesion with TSAM gave the surprising result that upon separation of the two beads under force, the ceramic broke rather than the TSAM. In summary, described and shown herein is that TSAMs can absorb the full force of supersonic impacts by basalt particles and larger pieces of aluminium shrapnel, providing the first example of a protein material capable of absorbing supersonic projectile impacts. These results lend the TSAMs towards use in sensory devices and incorporation into products that require the inclusion of a material with shock absorbing or sensory properties. It also has military applications, specifically as a backing for multi-layered armour where shattered ceramic capture is required, and in hypervelocity impact experiments in which the projectile needs to be preserved for further study. As a consequence of the endothermic energy dissipating mechanism of talin, it is very unlikely any elevation of projectile temperature was induced during the LGG experiment due to the induced endothermic process of protein unfolding (Schon, A. et al, (2017) Proteins-Structure Function and Bioinformatics 85, 2009-2016), offering a distinct benefit over aerogel materials. This energy dissipating mechanism of protein domain unfolding was confirmed using rheology, further evidenced by using GFP-VD1 to confirm that induced talin unfolding was the phenomenon responsible for the results observed in the rheological experiments. Through the refolding of talin domains within TSAM following the removal of force, potential reusability of TSAM is an additional property incurred if not directly penetrated. As talin contains thirteen helical domains, each with unique unfolding forces, these TSAMs may be tuneable by modifying the talin domains featured in the monomer unit offering the potential for tailoring toward a diverse array of mechanical properties and resulting applications.

Claims

CLAIMS: 1. A recombinant monomer comprising an amino acid sequence of, or derived from one or more rod domains of the protein talin, wherein the monomer comprises: - one or more helical structure; - at least one amino acid substitution in the monomer sequence; - N- and C-terminal sequence adaptations; and - means to enable cross-linking of the monomer and/or means for providing attachment to a cross-linking moiety.

2. The recombinant amino acid monomer according to claim 1, wherein at least one amino acid substitution is a cysteine/serine substitution.

3. The recombinant amino acid monomer according to claim 1 or claim 2, wherein the means to enable cross-linking is a coiled coil motif in the amino acid sequence and/or the means for providing attachment to a cross-linking moiety is a cysteine residue.

4. The recombinant amino acid monomer according to claim 3 where in the cysteine residue is at each or either of the N- and C-terminals.

5. The recombinant amino acid monomer according to any one of claims 1 to 4, wherein the amino acid sequence comprises sequences from consecutive rod domains.

6. The recombinant amino acid monomer according to claim 5, wherein the consecutive rod domain sequences are, or are derived from, domains R1, R2 and R3.

7. The recombinant amino acid monomer according to any one of claims 1 to 6, wherein the monomer comprises a sequence as set out in SEQ ID NO:2.

8. The recombinant amino acid monomer according to any one of claims 1 to 4, wherein the monomer comprises at least two repeats of, or derived from, an amino acid sequence of a single rod domain.

9. The recombinant amino acid monomer according to claim 8, wherein the single rod domain is, or is derived from, domains R1, R3, R9 or R13.

10. The recombinant amino acid monomer according to claim 8 or claim 9, wherein the single rod domain has a sequence of any one of SEQ ID NOs:3, 4, 5 and 6.

11. The recombinant amino acid monomer according to any one of claims 1 to 4, wherein the monomer comprises at least two repeats of, or derived from, an amino acid sequence of, or derived from, non-contiguous rod domains.

12. The recombinant amino acid monomer according to claim 11, wherein the non- contiguous rod domains are, or are derived from, R3 and R9.

13. The recombinant amino acid monomer according to claim 11 or claim 12, wherein the single rod domain has a sequence of SEQ ID NOS: 4 and 5.

14. The recombinant amino acid monomer according to any one of claims 8 to 13, wherein the repeats of the single or non-contiguous rod domain sequences are separated by a linker.

15. The recombinant amino acid monomer according to claim 14, wherein the linker has a sequence as set out in SEQ ID NO:7 or SEQ ID NO:8.

16. The recombinant amino acid monomer according to any one of claims 1 to 15, wherein all of the cysteine residues in the monomer sequence are substituted with serine.

17. The recombinant amino acid monomer according to any one of claims 1 to 16, wherein the N-terminal includes an adaptation having a sequence as set out in SEQ ID NO:9 or SEQ ID NO:10.

18. The recombinant amino acid monomer according to any one of claims 1 to 17, wherein the C-terminal includes an adaptation having a sequence as set out in SEQ ID NO:11 or SEQ ID NO:12.

19. The recombinant amino acid monomer according to any one of claims 1 to 18, wherein the monomer further includes an expression sequence.

20. The recombinant amino acid monomer according to claim 19, wherein the expression sequence has a sequence as set out in any one of SEQ ID NOs:13, 14 or 15.

21. The recombinant amino acid monomer according to any one of claims 1 to 20, wherein the monomer has an amino acid sequence comprising the sequence of any one of SEQ ID NOs:16 to 22.

22. A recombinant mechanosensory amino acid polymer in which the polymer comprises at least two amino acid monomer sequences and cross-linking means, characterised in that the monomer sequences include: one or more helical structure; at least one amino acid substitution; N- and C-terminal sequence adaptations; and means to enable cross-linking of the monomers and/or means for providing attachment to a cross-linker, wherein the monomers are cross-linked to form the polymer.

23. The recombinant mechanosensory polymer according to claim 22, wherein at least one of the monomers comprises, or is derived from, talin.

24. The recombinant mechanosensory polymer according to claim 22 or claim 23, wherein at least one of the monomers comprises, or is derived from, one or more rod domains of the protein talin as claimed in any one of claims 1 to 22.

25. The recombinant mechanosensory polymer according to any one of claims 22 to 24, wherein at least one amino acid substitution is a cysteine/serine substitution.

26. The recombinant mechanosensory polymer according to claim 25, wherein all of the cysteine residues in the monomer sequence are substituted with serine.

27. The recombinant mechanosensory polymer according to any one of claims 22 to 26, wherein means to enable cross-linking of the monomers is a coiled coil motif in the amino acid sequence and/or the means for providing attachment to a cross-linking moiety is a cysteine residue.

28. The recombinant mechanosensory polymer according to any one of claims 22 to 27, wherein the cross-linking means is an organic group capable of linking the monomers together through covalent interactions.

29. The recombinant mechanosensory polymer according to any one of claims 22 to 28, wherein the cross-linking means is a maleimide, optionally a tripoidal maleimide.

30. The recombinant mechanosensory polymer according to claim 29, wherein tripoidal maleimide has the structure of formula I: Formula I

31. The recombinant mechanosensory polymer according to any one of claims 24 to 26, wherein the cross-linking moiety is a Spy- or Avi-Tag.

32. The recombinant mechanosensory polymer according to any one of claims 22 to 31, wherein the polymer further comprises a marker or binding entity which changes properties of the polymer on unfolding and/or refolding of the helical structure.

33. The recombinant mechanosensory polymer according to claim 32, wherein the marker or binding entity imparts a change of colour, luminescence or fluorescence on either unfolding or refolding of the helical structure.

34. The recombinant mechanosensory polymer according to claim 32 or claim 33, wherein the marker or binding entity imparts a reversible or non-reversible change to the polymer when the helical structure is in an unfolded state.

35. The recombinant mechanosensory polymer according to any one of claims 32 to 34, wherein vinculin and a marker that includes one or more as motifs derived from KANK1-4, Deleted in Liver Cancer 1 (DLC1) and Rap1-GTP-interacting adaptor molecule (RIAM) proteins.

36. Use of a mechanosensory hydrogel comprising a recombinant mechanosensory monomer as claimed in any one of claims 1 to 21, or a recombinant mechanosensory polymer as claimed in any one of claims 22 to 35, to absorb, dissipate and/or sense, or to protect an object from kinetic energy or force applied to the hydrogel.

37. Use of a mechanosensory hydrogel comprising a recombinant mechanosensory monomer as claimed in any one of claims 1 to 21, or a recombinant mechanosensory polymer as claimed in any one of claims 22 to 35, to protect an object from a force or impact, including a microimpact, from or with another object.

38. Use according to claim 36 or claim 37, wherein the force is a mechanical shock from impact with an object.

39. Use according to claim 37 or claim 38, wherein the object is a high velocity, hypervelocity and/or explosive object such as a projectile, a bullet or space debris.

40. Use according to any one of claims 36 to 39, wherein the mechanosensory hydrogel additionally substantially retains the object within its structure.

41. Use of a mechanosensory hydrogel comprising a recombinant mechanosensory monomer as claimed in any one of claims 1 to 21, or a recombinant mechanosensory polymer as claimed in any one of claims 22 to 35, as an adhesive and/or coating for materials used in products designed to protect an object or person from a force or impact from or with another object.