WO2006051313A2

WO2006051313A2 - Peptides designed to switch folded state upon binding metals

Info

Publication number: WO2006051313A2
Application number: PCT/GB2005/004360
Authority: WO
Inventors: Derek Woolfson; Eleonora Cerasoli
Original assignee: University Of Sussex
Priority date: 2004-11-12
Filing date: 2005-11-14
Publication date: 2006-05-18
Also published as: GB0425033D0; WO2006051313A3

Abstract

A peptide having sequence and structural duality that can reversibly switch between a first structural form and a second structural form in response to an agent, wherein the peptide comprises interactive motifs that interact to form a bond in response to the agent and thereby cause the peptide to assume the second structural form, wherein the second structural form includes an agent binding site.

Description

Peptides

The present invention relates to peptides that switch between 2 structural states in response to the presence or absence of a stimulus. The present invention also relates to the use of the peptides of the present invention in an assay to detect the presence or absence of a stimulus.

Successful protein and peptide designs require reliable rules that relate protein sequence and structure. This is reflected in the bias towards predominantly α-helical, and, in particular, coiled-coil structures in successful designs made to date, which include: four-helix bundles, parallel dimeric and trimeric coiled coils and helix-turn-helix motifs. These early α-helical designs were backed by good experimental and theoretical models for σ-helical peptides, and the resulting development of rules for the folding of α-helical structures. Studies on the folding and design of ^-structures are also being pursued. Recently, however, the folding of small peptides into highly populated ^-structures in solution has been reported; and rules are being established that link sequence and /^-structure in proteins. .

Large conformational transitions within proteins, by which is meant large relative movements of, or changes in secondary structure, are increasingly being recognized as a means through which protein function (and dysfunction) is elicited and mediated. For example, in the serpins, which are suicide inhibitors of serine proteases, cleavage of the scissile bond leads to the transformation of a native loop into an internal strand of a β- sheet (Lee, Park et al, Natural Structural Biology, 3(6): 497-500, 1996). This conformational change cements interactions between the cleaved serpins and their targets. Peptides and proteins that rearrange to amyloid-like structures represent another type of transition in which ^-structure is formed (Rochet and Lansbury et al., Current Opinion in Structural Biology, 10(1): 60-60, 2000). Unfolded and folded polypeptides, which are not necessarily //-structured, transform to fibres rich in /?-sheet. The archetypal example of a conformational change involving the rearrangement of largely helical structure is the switching of influenza hemagglutinin into an active form competent for virus-host membrane fusion (Skehel and Wiley et al., Annual Review of Biochemistry, 69: 531-569, 2000). On a similar theme, the assembly of certain SNARE-protein complexes, which mediate the fusion of vesicle membranes, is inhibited by the native conformation of one component that must open prior to SNARE-complex assembly (Munson et ah, Nature Structural Biology, 7(101: 894-902, 2000, Tochio et ah, Science, 293(5530): 698-702, 2001). Transitions in structures that are accompanied by changes in protein oligomer state and, in turn, protein function include the release and assembly of the trimerisation region and subsequent DNA binding of certain heat shock transcription factors (Rabindran et ah, Science, 259: 230- 234, 1993) and the dimer-tetramer switch in IFi, which regulates the mitochondrial ATP synthase (Cabezon et ah, EMBO Journal, 20(24): 6990-6996, 2001). International patent application WO 99/11661 discloses a protein comprising an elastomeric peptide which increases in length in response to an increase in temperature; however, there is no overall change in the structure of the protein. Finally, pH changes and proximity to membranes triggers certain colicin domains to switch from water-soluble globular structures to integral-membrane proteins, which then switch between an open and closed channel state depending on an applied membrane electrical potential.

In other cases, conformational changes appear to be abnormalities, or misfolding events and result in disease. For example, in Alzheimer's dementia and prion diseases a-to-β structural transitions in peptides and proteins are implicated in the formation of amyloid fibrils and the pathogenesis of disease. Jarvet et ah, (J. Am. Chem. Soc, 122, 4261- 4268, 2000) discusses such structural changes in the Alzheimer peptide fragment Ab(12-28).

The examples given above may be regarded as proteins with internal structural conflicts; that is, protein sequences with different accessible folded states and the form that is expressed depends on the prevailing conditions. This appears to run against Anfmsen's hypothesis that proteins adopt the thermodynamically most-stable state (Anfmsen et ah, Science, 181: 223-230, 1973). This idea is not new, for example, the cross-/? structure of amyloid fibres has long been considered the true energy minimum for all polypeptide chains; native influenza hemagglutinin is described as a spring- loaded, metastable conformation that is trapped awaiting a trigger to switch to the fusogenic form (Carr et ah, PNAS USA, 94(26): 14306-14313, 1997); the native serpins are referred to as stressed states, cleavage of which by the targeted protease brings about a conformational transition to the relaxed state (Lee, Park et al, Nature Structural Biology 3{6): 497-500, 1996, Whisstock et al, Trends in Biochemical Sciences, 23: 63-67, 1998); and the inactive forms of certain SNAREs are referred to as "closed" conformations in which the SNARE oligomerisation motif is rendered inaccessible (Munson et al, Nature Structural Biology, 7ClO): 894-902, 2000, Tochio et al, Science, 293(5530): 698-702, 2001).

One way to consider designing conformational switches is to set up a structural conflict within a peptide or protein by superimposing motifs for two different structures in a single sequence, hi this way, Anfmsen's basic tenet of protein folding is not contravened, it is simply that one sequence motif will be frustrated when the alternative structure is adopted. The inventors refer to such polypeptides as having sequence and structural duality. The inventors introduced and tested this concept previously. In particular, Ciani et al (J. Biol. Chem., 277, 10150-10155, 2002) designed peptides that switched from a α-helical structure to a ^-helical structure. It was only possible to reverse the conformation of the peptides from the β structure (amyloid like fibrils) to the α-helical structure by cooling the sample, lowering its pH to pH 2 and waiting a number of days. Such a change would be impractical and, therefore, more or less useless in any embodiments of switching peptides in diagnostics, etc.

The Abstract from Japanese Patent Application JP- A-7157499 discloses a peptide that is indicated as reversibly switching between an α-helical structure and a /?-sheet structure. No data is provided showing reversible switching and the sequence of the peptide is not disclosed.

Zhao et al, (Protein Science, IJ), 1113-1123, 2001) discloses that the yeast adhesion protein α-agglutinin can reversibly switch from a β rich structure to a mixed alβ structure by changes in temperature or pH. Reed et al, (Biochemistry, 30, 4521-4528, 1991) discloses that peptides derived from the CD4 binding domain of gpl20 switch from an α-helical structure to a /?-sheet structure following changes in the polar conditions. The papers provide no information concerning obtaining or designing other switching peptides. Ciani et al., describes a heat induced switch from an α-helix to /?-sheet structure.

Micklatcher and Chmielewski describe an α to β transition in "Helical peptide and protein design" Current Opinion in Chemical Biology, vol 3, 1999, p724 to 729 but do not provide switches that respond reversibly to a stimulus.

International Patent Application WO2004/020464 describes peptides that are able to switch between two structural states in response to the presence or absence of a stimulus. In particular, this document describes peptides that can change conformation in response to oxidation or reduction. The peptides described switch between a parallel coiled-coil dimer and an anti-parallel coiled-coil monomer.

There is a requirement for a peptide that can reversibly switch between two different structural conformations in response to the presence or absence of other stimuli, in particular the presence of agents, such as metal ions. It is also desirable that the peptide can switch between the two different structural conformations without the requirement of having to denature the protein by heating, or irreversible chemical actions.

It is particularly desirable to be able to test for the presence or absence of zinc, especially because of zinc's biological roles. The use of zinc sensors including zinc fingers has already been investigated, but such sensors have had significant problems with oxidation and digestion by proteases.

In the present invention there is provided a peptide having sequence and structural duality which can reversibly switch between a first structural form and a second structural form in response to an agent, wherein the peptide comprises interactive motifs that interact to form a bond in response to the agent and thereby cause the peptide to assume the second structural form, wherein the second structural form includes an agent binding site.

The peptide of the present invention has sequence and structural duality so that the peptide can assume two different structural forms and reversibly switch between the forms in response to the presence or absence of an agent. The peptide can therefore be used to detect the presence or absence of the agent and monitor for changes in the level of the agent because the peptide can reversibly switch between the two structural forms.

The peptide can switch between the different structural forms without the need to denature the protein by heating or chemical reactions, e.g. reducing the pH to pH 2. Furthermore, the switch between structural forms also occurs relatively quickly, e.g. in less than one or two hours.

The term agent includes any atom or molecule that can bind to the peptide. In particular it refers to metal ions. Preferred metal ions include zinc, calcium, cobalt. Preferably the agent is not a hydrogen ion. The agent binding site may be any site that allows an agent to bind. The agent binding site is preferably specifically designed to correspond to the agent. The agent binding site is not a disulphide bond. The terms agent and agent binding site would be understood by those skilled in the art.

It is particularly preferred that the agent is a zinc ion, and that the agent binding site is a zinc binding site.

The term "peptide" as used herein refers to a polymer of amino acids. The peptide can be of any length but is preferably between about 20 and 400 amino acids in length. It is further preferred that the peptide is between about 30 and 200 amino acids in length.

The term does not refer to or exclude post-expression modifications of the peptide, for example, glycosylations, acetylations and phosphorylations. Included in the definition are peptides containing one or more analogues of an amino acid, including unnatural amino acids.

The term "reversibly switch" is used to indicate that the peptide can reversibly switch between the first and second structural forms. Therefore the peptide can switch from the first structural form to the second structural form and back again. As indicated above the majority of prior art peptides that are capable of switching structure can only switch from a first structure to a second structure but not back again. Such prior art peptides cannot be reused and cannot be used to monitor changes in the level of a stimulus because once the peptide assumes the second structure it is no longer responsive to the stimulus. The term "sequence and structural duality" means that the peptide has a sequence that is compatible with 2 different structures. Generally, the peptide has 2 different superimposed sequence and structural motifs enabling the peptide to assume two different structures, namely a first and a second sequence and structural motif. Preferably the first structural form is a parallel coiled-coiled oligomer structure especially a dimer or trimer, most preferably a trimer. Preferably the first sequence motif of the peptide is a coiled-coil motif for a parallel coiled-coil oligomer structure especially a dimer or trimer, most preferably a trimer.

The second structural form includes an agent binding site. The presence of the agent causes the peptide to assume the second structural form, as the agent binds to the agent binding site. Preferably the agent binding site is a metal ion binding site. More preferably it includes a zinc binding site. Most preferably, the second structural form is a zinc finger structure.

To bring about the second structural form, the second sequence motif preferably includes a motif for an agent binding site, preferably a metal ion binding site. Preferably the motif is a motif for a zinc binding site. Most preferably, the second sequence motif is a motif for a zinc finger.

The term "zinc finger" is well known to those skilled in the art. A zinc finger is a peptide structure which has a finger-like configuration, brought about by the binding of the peptide to zinc.

The first and second structural forms of the peptide according to the present invention can be any structural forms provided they are sufficiently different from each other to enable one skilled in the art to easily determine if the peptide is in the first structural form or the second structural form, and preferably to measure the ratio between the first and second structural forms of the peptide. Preferably, the first structural form of the peptide oligomerises and the second structural form of the peptide is a monomer. This is advantageous because it is relatively easy to distinguish between an oligomer and a monomer. Suitable methods of determining if the peptide is in the first or second structural form include circular dichroism spectroscopy and non-denaturing PAGE. Split proteins and enzymes may also be used to detect the conformational change of the protein (see Ghosh et al., J. Amer. Chem. Soc, 122, 5658-9, 2000; Johnsson et al., PNAS USA,_9_L 10340-4, 1994, and Hocker et al., Nat. Struct. Biol., 8, 32-36, 2001).

Preferably, the first structural form is a continuous helical structure. As indicated above, the first structural form can preferably oligomerise. The peptide in the first structural form may form a heteroligomer or a homooligmer. It is particularly preferred that the peptide in the first structural form is a parallel coiled-coil trimer.

The term "interactive motifs" as used herein means any motifs that can interact with each other or with an agent to form a covalent or non-covalent bond or bonds in response to the agent. Preferably the interactive motifs interact with the agent form a non-covalent bond or bonds. By the interactive motifs forming a bond the peptide of the present invention is caused to assume the second structural form.

The interactive motifs may be adjacent to each other and positioned as a single unit in the peptide, hi the present invention, the interactive motifs are part of an agent binding site, preferably a metal binding site. The interactive motifs may be positioned within the peptide as a single unit. Alternatively, the interactive motifs can be in separate parts of the peptide. The interactive motifs can be at any position in the peptide provided they can interact with each other or the agent to form a bond or bonds. Preferably, the interactive motifs are in separate parts of the peptide, that is to say the interactive motifs are not adjacent to one another. Preferably, the interactive motifs are near the ends of the peptide.

The interactive motifs comprise parts of an agent binding site, preferably a metal binding site, that form a non-covalent linkage in response to the presence of the corresponding metal ion. Suitable binding sites are known to those skilled in the art. A part of the binding site can be incorporated into one part the peptide of the present invention and a second part of the binding site can be incorporated into a second part of the peptide of the present invention, wherein when the agent, preferably a metal ion, is present, the parts of the binding site interact via the agent to form a bond or bonds.

Preferably the interactive motifs are kept separate from any amino acids that are important in the first structural form. In particular, where the first structural form is a coiled-coil, the interactive motifs are kept away from the coiled-coil interface. It is particularly preferred that the peptide has a non-canonical coiled-coil pattern of hydrophobic (h) and polar (p) or other residues, especially arranged as follows:

phpphppphppphpphppphpphppph.

In a first particularly preferred embodiment of the present invention the peptide according to the present invention comprises the following sequence:

abcdefghijka 'b 'c 'd 'ef ' 'g 'a"b"c"d"e"fg"

wherein: a 'b 'c 'd 'ef ' 'g ' and a"b"c"d"e"fg" are heptad sequence motifs; abcdefghijk is a hendecad sequence motif; and wherein b, e, g' and/' form an agent binding site.

The term "heptad sequence motif refers to any seven amino acid motif which is capable of forming a helical coiled-coil structure. Suitable heptad repeat sequences are well known to those skilled in the art.

The term "hendecad sequence motif refers to any eleven amino acid motif which begins with a heptad motif and which is capable of forming a helical coiled-coil structure. Suitable hendecad repeat sequences are well known to those skilled in the art.

Heptad and hendecad sequence motifs are known in the art and are described in WO2004/020464.

Preferably d, d' and d" are leucine. It is further preferred that a, a ' and a" are isoleucine.

It has been found that by restricting the a, a ', a ", d, d' and d" residues as indicated above, heptad and hendecad repeats are formed that code particularly well for helix coiled-coil structures. It is also preferred that b and e are histidine or cysteine. Most preferably both are histidine. This reduces problems with oxidation associated with the presence of cysteine.

In addition, g ' is preferably histidine, as is/".

These amino acids form the zinc binding site, causing the formulation of the zinc-finger structure in the presence of zinc.

It can be seen that the amino acids forming the zinc binding site, namely b, e, g" and/' are kept separate from the amino acids involved in forming the coiled-coil, namely a, a\ α", d, d' and d '.

Preferably, the peptide of the present invention comprises the following sequence: IHALHRKAFAKIARLERHIRALEHA

One or more additional amino acids may be present at one or both ends of the peptide.

In particular, the peptide of the present invention comprises the following sequence: YIHALHRKAFAKIARLERHIRALEHAA

In addition, the peptide may have an acetyl group at the N terminal and an NH₂ group at the C terminal, as follows: ac-YIHALHRKAFAKIARLERHIRALEHAA-am

In a second preferred embodiment, the peptide comprises the sequence:

abcdefgabcdefsa 'b 'c 'd 'e Jg 'a 'b 'c 'd'e'f'e',

wherein abcdefg, abcdefg, a 'b 'c 'd 'ef ' 'g ' and a 'b'c'd'e'f'g' are heptad sequence motifs, and c, a, g ' and£ form the agent binding site.

Preferably d, d, d' and d/_ are leucine. Preferably αj is isoleucine, and preferably a ' is asparagine. Asparagine is preferably present for dimer specification.

a is preferably tyrosine, and is included as a chromophore.

Preferably c, a, g' and/' are histidine. These amino acids form a zinc binding site.

It is particularly preferred that the peptide comprises the sequence

YGHLEQKHAFLEQKNAALEQHIRALEHK

One or more additional amino acids may be present at one or both ends of the peptide,

hi particular, the peptide of the present invention comprises the following sequence:

KYGHLEQKHAFLEQKNAALEQHIRALEHKIRKLE

In the second sequence the interactive motifs, namely the histidine residues that form the zinc binding site overlap at position a with the residues responsible for providing the coiled-coil structure. The other histidine residues are separate from the coiled-coil interface. The inventors surprisingly found the a position in the heptads {a, a, a ' and α') to be tolerant of substitutions, but found that the d position (d, d, d' and dj was intolerant of substitution.

As will be apparent to those skilled in the art modifications of the sequence of the peptides of the present invention can be made to alter the peptides characteristics. For example, modifications can be made to the peptide so that it requires a greater presence of ligands to switch conformation, thereby enabling the peptide to be used to detect a high level of the agent. Other modification, well known to those skilled in the art, can be made to the peptide to increase its solubility, etc.

The present invention also provides the use of the peptide for determining the presence or absence of an agent.

The present invention provides a method of detecting an agent comprising incubating a peptide of the present invention with a test solution, and determining if the peptide has the first structural form or the second structural form in the test solution, wherein if the peptide assumes the second structural form, the test solution contains the agent. The change in the structure of the peptide can be measured using any technique. Suitable techniques include circular dichroism spectroscopy, fluorescence quenching, Fluorescence Resonance Energy Transfer (FRET), surface plasmon resonance, mass spectrometry and non-denaturing PAGE (particularly where the peptide oligomerises in only one structural form). Split proteins and enzymes may also be used to detect the conformational change of the protein (see Ghosh et ah, J. Amer. Chem. Soc, 122. 5658-9, 2000; Johnsson et al, PNAS USAJH, 10340-4, 1994, and Hocker et al, Nat. Struct. Biol., 8, 32-36, 2001). In the case of FRET, donor and acceptor fluorophores can be added to parts of the peptide so that they are only brought into proximity with each other in one of the conformational states.

The present invention is now described by way of example only with reference to the following drawings.

Figure 1 shows size-exclusion chromatography for the peptide (ZiCo) without (broken line) and with (solid line) zinc.

Figure 2 shows FT-IR second derivative (left) and CD spectra (right) for the peptide (ZiCo) without zinc in phosphate buffer (dashed line) and with zinc in Tris buffer (solid lines).

Figure 3 shows zinc binding in Tris buffer followed by CD spectroscopy: left, without (broken line) and with zinc (solid line); right, as on the right, but without (solid line) and with added EDTA (broken line).

Figure 4 shows the CD melting curve for 100 μM peptide (ZiCo) in Tris with Zinc.

Figure 5 shows zinc titration into peptide (ZiCo) in Tris Buffer.

Figure 6 shows semi-preparative RP-HPLC of crude ZiCo peptide.

Figure 7 shows analytical RP-HPLC of purified ZiCo. Figure 8 shows MALDI-TOF mass spectrometry of the main peak from the RP-HPLC. The expected mass of ZiCo is 3231.8 Da, and the calculated mass of the main peak from RP-HPLC was 3233.3± 1.0 Da.

Figure 9 shows infrared (A, B) and second derivative spectra (C, D) of ZiCo in the absence (A, C) and in the presence (B, D) of zinc. In the absence of zinc (A, C), there is a main band at 1649 ± 2 cm^"1 that can be assigned to α-helical structure and a small component at 1680 ± 2 cm^"1 that can arise from the fraying of the termini of the helix, hi the presence of zinc (B, D), additional bands are present, notably the band at 1633 ± 2 cm^"1 can be assigned to /?-sheet structure.

Figure 10 shows analytical ultracentrifugation data of ZiCo (A) without zinc, and (B) with zinc. The bottom panels show sedimentation equilibrium data recorded on ZiCo as plots of absorbance versus radial position (r /2, cm²) at three speeds for a given protein concentration at 5 °C. A: 25,000 rpm (circles), 30,000 rpm (squares), 36,000 rpm (triangles). B: 36,000 rpm (circles), 43,000 rpm (squares), 56,000 (triangles). The fits of these data sets to a model incorporating a monomer-trimer equilibrium (A) or a single species model (B) are also shown. The three upper panels illustrate the residuals for the fits to the three data sets.

Figure 11 shows titration curves. The left and right panels refer to different protocols for titration. The curve on the left panel was obtained by titrating zinc in a cell containing a solution of the peptide (Protocol I); the titration on the right panel was obtained by subtracting the heat of the coiled-coil dissociation (ZiCo in the syringe and buffer in the cell) from the heat of the titration of a concentrated ZiCo solution into buffer with zinc (Protocol II).

The drawback of the protocol I is the high heat of dilution due to the impossibility of match buffers in the syringe (ZnCl₂ has to be prepared in water because Zn₃(PO4)₂ is very insoluble) and in the cell. The drawback of the protocol II is, instead, the composite nature of the binding process (coiled-coil dissociation and zinc binding). The protocols are as follows: Protocol I Protocol II

N 0.968 ± 0.0105 0.523 + 0.00956 K_d 4.8 + 0.4 μM 2.9 ± 0.3 μM ΔH -10.75 ± 0.187 IcCaI mOr¹ -10.31 + 0.253 IcCaI mOr¹ ΔS -13.7 cal K-^l mor' -l l.l cal K-' mol-¹

Figure 12 shows ID ¹H NMR spectra of 100 μM ZiCo, 50 niM sodium phosphate, pH 7.5, (A) without, and (B) with zinc.

Figure 13 shows CD spectra for ZiCo without (solid line) and with (dashed line) zinc. Conditions: 100 μM peptide, 5 ⁰C, 50 niM sodium phosphate, pH 7.5, 50 mM NaCl; and with 100 μM ZnCl₂ (dashed spectra).

Figure 14 shows thermal unfolding curves for ZiCo. (A) Without zinc at 50 μM (line), 100 μM (dashes) and 200 μM (dashes and dots) peptide. (B) With equimolar zinc at 50 μM (line) and 100 μM (dashes) peptide.

Figure 15 shows AUC data for ZiCo: (A) 400 μM peptide, 30,000 rpm; (B) 150 μM peptide with equimolar zinc, 36,000 rpm. Experimental data are shown as open circles, and theoretical curves for monomers, dimers and trimers as broken, gray, and black lines, respectively. Conditions: 5 ⁰C 50 mM sodium phosphate, pH 7.5, 50 mM NaCl.

Figure 16 shows zinc binding (black squares) and EDTA-induced release (gray circles) by ZiCo monitored by CD spectroscopy. Conditions: 5 ⁰C 50 mM sodium phosphate, pH 7.5, 50 μM peptide (for both titrations).

EXAMPLES

Materials and Methods

Peptide synthesis.

Peptides were made on a Pioneer Peptide Synthesis System (Perseptive Biosystems) using standard Fmoc chemistry. They were purified by reversed-phase HPLC and their identities confirmed by MALDI-TOF mass spectrometry. Purified peptides were stored at pH 2, -20 ⁰C, and concentrations were estimated by UV absorption at 280 nm (e =1490 M^"1 cm^"1). Oxidised peptides were prepared by agitation of a 100 mM peptide solution at room temperature overnight in 0.1 M Tris-HCl pH 8.5 containing 6 M guanidine hydrochloride. Alkylated peptides were prepared by (1) incubation of a 1 mM peptide solution at 40 ⁰C for 1 h in 0.6 M Tris-HCl pH 8.6 containing 1.25 % (v/v) 2-mercaptoethanol, 8 M urea, 5 mM EDTA; (2) mixing the resulting reduced peptide solutions with 0.75 ml fresh 0.36 M iodoacetamide solution and incubating at room temperature for 15 min in the dark; and (3) dialysis against water. Chemical modification of cysteines was confirmed by MALDI-TOF mass spectrometry.

Circular dichroisni spectroscopy. CD measurements were made using a JASCO J- 715 spectropolarimeter fitted with a Peltier temperature controller. Peptide solutions were prepared in potassium phosphate, sodium phosphate or Tris buffers at pH 6 to 8 (5OmM) or 5OmM Nace and were examined in 1 mm or 1 cm quartz cuvettes. Spectra were recorded at 5 ⁰C using 1 nm intervals, a 1 nm bandwidth and 4, 8 or 16 sec response time. After base line correction, ellipticies in mdeg were converted to molar ellipticies (degree cm² dmol^"1) by normalising for the concentration of peptide bonds and path length. Thermal unfolding curves were recorded at 222 nm through 1 °C min^" ¹ ramps using a 1 or 2 nm bandwidth, averaging the signal for 16 s every 1 ⁰C intervals. Several methods were used to estimate the midpoints (Tms) of these curves, notably, taking first and second derivatives of the curves and fitting the curves to sigmoid functions.

Analytical Ultracentrifugation. Sedimentation equilibrium experiments were conducted at 5 ⁰C in a Beckman-Optima XL-I analytical ultracentrifuge fitted with an An-60 Ti rotor. 100 μl peptide solutions at 150-600 μM in 50 mM sodium phosphate buffer, pH 7.5, 50 mM NaCl, without or with equimolar zinc were equilibrated at speeds in the range 25,000 to 44,000 rpm. Data were fit simultaneously assuming either a single, ideal species model, or a monomer-trimer equilibrium model in NONLIN (Johnson, M. L.; Correia, J. J. Biophys. J. 1981, 36, 575-588). Data simulations were prepared in the Beckman-Optima XL-AJXL-I data analysis software (v6.03). The monomer molecular weight (3231.8 Da) and partial specific volume (0.7456) of ZiCo were calculated from the amino-acid sequence, and the viscosity of the buffer at 5 ⁰C was taken to be 1.008 nig ml^"1 (Hayes, D. B.; Laue, T.; Philo, J. Sednterp 1995-1998, University of New Hampshire, U.S.A.).

FT-IR. ATR-FT-IR spectra were acquired on a Tensor 27 (Bruker Optics). Spectra were corrected for water vapour using the "atmospheric compensation" function provided by the Opus software (Bruker), followed by subtraction of the buffer spectrum. Gaussian fitting analysis was performed using the "curve fitting" macro with a bandwidth of 6.5 and resolution enhancement factor of 3. As starting points for the fitting were used peak maxima calculated by the second derivative of the spectrum (9 point smoothing, Savitzky- Golay function).

Isothermal titration calorimetry.

Measurements were obtained using a VP-ITC Microcal instrument using two different protocols.

Protocol I: a concentrated ZnCl₂ solution in H₂O (5 mM) was titrated into the cell compartment containing 100 μM ZiCo, 50 mM sodium phosphate, pH 7.5, 50 mM NaCl. The same zinc solution in H₂O was titrated into buffer without ZiCo and the heat of dilution was subtracted from the titration data before further analysis.

Protocol II: a solution of 0.5 mM ZiCo in 50 mM sodium phosphate, pH 7.5, 50 mM NaCl was titrated in the same buffer containing 50 μM ZnCl₂. To take the heat of coiled-coil dissociation into consideration, a titration was carried out adding 0.5 mM ZiCo into buffer without zinc. The heat of dissociation obtained in this way was first corrected for the heat of buffer dilution (titration of buffer against buffer) and subsequently subtracted to the titration data before analysis. Data were fitted to a one- binding-site model provided by Microcal ITC extension (Origin software).

NMR spectroscopy. Samples for NMR contained 100 μM ZiCo, 50 mM sodium phosphate, pH 7.5, 10% D₂O, either without or with 100 μM ZnCl₂. ID ¹H NMR spectra were recorded at 5 ⁰C on a Varian Inova600 spectrometer, equipped with a 5mm HCN triple resonance probe and z-axis pulsed field gradients. For the design the inventors used as starting points two well studied folds: the dimeric coiled coil and the classical zinc finger fold (Berg J.M., Godwin H. A. Annu. Rev. Biophys. Biomol. Struct., 1997, 26: 357-371). These structures are well known to those skilled in the art. The inventors have superimposed on the same sequence characteristics of both: the peptide was designed to adopt, in the absence of zinc, a coiled coil conformation and, in the presence of Zn²⁺, a zinc finger fold. To reach this goal the consensus sequence for the classical zinc fingers (Cys2 His2: (F/Y) X C X_2-4 C X₃ F X₅ L X₂ H X_3-5 H X _2-6) was superimposed onto heptads repeat patterns typical of the coiled coils. The designed peptide was named ZiCo (Zinc finger- Coiled coil).

The rationale is that the zinc finger fold seems to be highly dependent on the positioning of a few amino acids, namely, those of the consensus sequence. This has been demonstrated by the synthesis of a "minimalistic" zinc finger where all the amino acids, a part from those of the consensus sequence, were mutated to Alanine and Lysine (this last one used to improve the solubility).

For the design we kept the positions of the amino acids in the zinc finger consensus sequence and we made this compatible with the coiled coil requirements.

The amino acid sequence of a coiled coil is characterized by heptad repeats (abcdefg) in which hydrophobic residues in a and d positions form the hydrophobic core of the dimer with a 3-4 periodicity. The first step in the design was to compare the abcdefg pattern of the coiled coil with the zinc finger consensus sequence to find a alignment. In the coiled coils He and Leu residues in a and d positions direct the formation of the parallel dimer. For this reason the inventors took the Leu in the zinc finger consensus sequence as an anchor fixing this position as a "d" position of the coiled coil. Then the inventors started to fill the gaps left with positioning, where possible, He and Leu in the proper positions and taking care to put the zinc binding residues away from the coiled coil interface to avoid metal binding in the coiled-coil conformation. In the final design the first two Cys of the classical zinc finger motif (Cys2His2) were replaced with His to avoid problems connected with Cys oxidation. This was found to be compatible, at least in one case with the formation of the zinc finger. In this first design the 11-7-7 (hendecad, heptad, heptad) pattern was chosen because in zinc fingers the turn occurs around the 1 l-12^th aa. An 11 residue insert was used in WO2004/020464 is thought to give more flexibility to this part of the coiled coil structure (unwinding) whilst remaining compatible with the coiled coil dimer.

The best superimposition between the 11-7-7 pattern and the zinc fingers consensus sequence was found to be:

gabcdefghijkabcdefgabcdefga

YXCXXCXXXFXXXXXLXXHXXXXXHXX ac-YIHALHRKAFAKIARLERHIRALEHAA-am ZiCo

The inventors checked that the presence of the hendecad did not affect the coiled coil structure of the peptide, by checking the structure in a benign medium such as phosphate buffer.

In this buffer the peptide was folded as a coiled-coil oligomer. The CD spectra and melting curves were concentration dependent as expected for an oligomerising system. Moreover size-exclusion chromatography (SEC) showed the presence of two species in solution. Analytical Ultracentrifugation confirmed the existence of an equilibrium between monomer and dimer. FT-IR spectra showed only a single band in a position indicative of α-helical structure.

To test the design it was necessary to titrate with zinc. Unfortunately because of the low solubility of zinc phosphate the inventors could not perform such titrations in phosphate buffer. For this reason the buffer was changed to Tris-HCl. In these conditions ZiCo is less folded as a coiled-coil. The second derivative of the FT-IR spectra showed a peak at an intermediate position between the one for an unfolded structure and one for an α- helical structure. As a control EDTA was added to ZiCo in both phosphate buffer and in Tris-HCl in the absence of zinc. The addition of EDTA did not have any effect on the intensity of the circular dichrosim spectrum, indicating that the structure observed is not due to residual zinc present in the buffers. Upon zinc addition in Tris-HCl there was further re-folding of the peptide. This can be seen by CD spectroscopy. The FT-IR spectra showed the appearance of a band indicative of the formation of /?-sheet structure.

AU the experiments for the characterization of the coiled-coil conformation were carried out in phosphate buffer, all the experiments for the characterization of the folded conformation in the presence of Zinc were carried out in Tris-HCl.

ZiCo in Tris buffer binds zinc and adopts a monomelic fold. Size exclusion chromatography run in the buffer favouring the formation of the zinc-finger fold shows only one main sharp peak indicating the presence of a monomelic folded species in solution.

Zinc binding to ZiCo, as for other zinc finger peptides, is reversible. Additions of EDTA shift the equilibrium back though removal by chelation of the bound zinc.

Moreover Zinc binding is specific. Though more experiments are necessary to investigate ZiCo specificity at a broader range of metals and at various concentrations, preliminary experiments showed that no folding is determined by equimolar concentrations of nickel, cobalt or copper (data not shown).

To confirm the results, further experiments were carried out.

ZiCo was synthesized by Fmoc-based solid-phase methods, purified by RP-HPLC and confirmed by MALDI-TOF mass spectrometry. Circular dichroism (CD) spectra of ZiCo without Zn²⁺ showed minima at 208 and 222 run indicative of α-helix (Figure 2). Consistent with this, the FT-IR spectrum had a single band at 1651 ± 2 cm^'1 . The intensity of the CD signal at 222 nm suggested ~ 50% helix.¹³ This structure unfolded with a sigmoidal curve upon heating indicative of a cooperatively folded structure, albeit of low thermal stability: the midpoint of the unfolding was 14±1 ⁰C at 100 μM peptide as calculated by method of first derivative. As expected for a coiled-coil oligomer, the T_M was concentration dependent: at 50 μM peptide the TM was lO±l ⁰C and 18±1 ⁰C at 200 μM peptide. Analytical ultracentrifugation (AUC) data gave a molecular weight of 8,510±461 Da when fitted to a single-species model, and a KQ of 10-100 IM with a monomer-trimer equilibrium model. Together, these data are consistent with ZiCo forming an α-helical coiled-coil oligomer in the absence of zinc as designed.

Upon the addition of zinc, both the CD and FT-IR spectra changed: the intensity of the

CD signal increased indicative of an increase in helical content of -15%; and the FT-IR spectra had an additional component at 1633 ± 2 cm^"1 indicative of b-sheet. The thermal unfolding behavior also changed: firstly, zinc stabilized the structure. Secondly, with zinc the TM of ZiCo was concentration independent at 23±1 ⁰C consistent with the peptide folded as a monomer as desired. This was confirmed by AUC, which fitted best to a single species model with a molecular weight of 3,142±328 Da in reasonable agreement with the predicted monomer molecular weight with zinc of 3,296 Da.

To investigate zinc binding further, and to check for reversibility, CD spectroscopy was used to follow zinc titrations at 50 μM and 100 μM peptide concentrations. In both cases, saturation was achieved at equimolar concentrations of peptide:Zn²⁺, and no further changes were observed upon excess Zn²⁺ addition.¹⁴ Furthermore, the switches were reversible as the signal returned to that of the peptide alone upon addition of EDTA. The non-coincidence of the binding and release curves indicated the expected higher affinity of EDTA for zinc, which is reported as 10^"16 M.^8e The binding affinity of ZiCo was measured directly by isothermal titration calorimetry (ITC): the titration curves fitted to single-site binding models and returned KpS in the range 3 - 5 μM.

A difference in structure of the two forms of ZiCo was further supported by ID ¹H NMR spectra of ZiCo without and with Zn²⁺. Spectra recorded without Zn²⁺ showed little dispersion consistent with an all α-helical fold without buried aromatic residues. However, those with zinc present showed greater chemical shift dispersion and sharper peaks consistent with a smaller alternately folded species. We are pursuing high- resolution structural studies of both forms. In summary, we describe the design and solution-phase characterization of a peptide that reversibly switches between a oligomeric, particularly trimeric α-helical coiled coil, and a zinc-bound folded monomer. Given the important biological role of zinc and interest in metal sensing for practical applications, further development of the ZiCo system could render a novel zinc sensor.

The inventors have produced a system in which addition of a agent causes a switch between two different structures. The characteristic of both structures are integrated in one sequence.

The zinc ion has particular biological roles and so the development of useful zinc sensors has attracted much interest and different approaches have been used for their development (Kimura E.; Aoki S. BioMetals 2001, 14, 191-204). The use of zinc fingers as sensors has been already investigated. One of the problems connected with the use of these systems is the vulnerability to air oxidation and the digestion by proteases.

The peptide of the invention is not vulnerable to air oxidation because of the substitution of His residues for Cys. In addition, unlike previously described sensors and systems, the peptide is a switch and not a folding process. The peptide is more resistant of proteolysis than previously described folding processes. Also, the peptide allows a better dynamic range of reporter signals (e.g. fluorescence) to be engineered.

AU documents cited herein are incorporated by reference.

Claims

1. A peptide having sequence and structural duality that can reversibly switch between a first structural form and a second structural form in response to an agent, wherein the peptide comprises interactive motifs that interact to form a bond in response to the agent and thereby cause the peptide to assume the second structural form, wherein the second structural form includes an agent binding site.

2. The peptide according to claim 1, wherein the first structural form of the peptide can oligomerise.

3. The peptide according to claim 2, in which the first structural form of the peptide can oligomerise but the second structural form cannot oligomerise.

4. The peptide according to claim 1, 2 or 3, wherein the agent binding site is a metal binding site and the agent is the metal.

5. The peptide according to claim 4 wherein the metal binding site is a zinc binding site, and the metal is zinc.

6. The peptide according to any preceding claim, wherein the peptide forms a continuous helical structure as the first structural form.

7. The peptide according to claim 2 or 3, wherein the first structural form of the peptide trimerises to form a parallel coiled-coil trimer.

8. The peptide according to any preceding claim, wherein the peptide forms a zinc finger structure as the second structural form

9. The peptide according to any preceding claim, wherein the peptide has two different superimposed sequence and structural motifs.

10. The peptide according to claim 9, wherein the first sequence motif is a coiled-coil motif for a parallel coiled-coil oligomer structure.

11. The peptide according to claim 9 or claim 10, wherein the second sequence motif is a zinc finger motif.

12. The peptide according to any one of the preceding claims, wherein the interactive motifs form a covalent or non-covalent bond in response to the presence of the agent.

13. The peptide according to claim 12, wherein the interactive motifs form the agent binding site.

14. The peptide according to claim 13, wherein, in the presence of the agent, the interactive motifs form a bond with the agent.

15. The peptide according any preceding claim, comprising the following sequence: abcdefghijka 'b 'c 'd 'ef ' 'g 'a"b"c"d"e"fg"

wherein: a 'b 'c 'd 'ef ' 'g ' and a"b"c"d"e"f'g" are heptad sequence motifs; abcdefghijk is an eleven heptad sequence motif; and wherein b, e, g" and/' form the agent binding site.

16. The peptide according to claim 15, wherein d, d^* and d" are leucine.

17. The peptide according to claim 15 or 16, wherein the a, a' and α" are isoleucine.

18. The peptide according to any one of claims 15 to 17, wherein b and e are cysteine or histidine.

19. The peptide according to claim 18, wherein b and e are histidine.

20. The peptide according to any one of claims 15 to 19, wherein g is histidine.

21. The peptide according to any one of claims 15 to 20, wherein/" is histidine.

22. The peptide according to any one of claims 15 to 21, wherein the peptide comprises the following sequence:

fflALHRKAFAKIARLERHIRALEHA.

23. The peptide according to any of claims 1 to 14, wherein the peptide comprises the following sequence: abcdefgabcdefsa 'b 'c 'd 'e fg 'a 'b 'c'd'e'fz'.

wherein abcdefg, abcdefs. a 'b 'c'd'ef' 'g' and a 'b 'c'd'e'f's' are heptad sequence motifs, and c, a, g ' and/' form the agent binding site.

24. The peptide according to claim 23, wherein d, d, d' and (T_ are leucine.

25. The peptide according to claim 23 or 24, wherein q!_ is isoleucine.

26. The peptide according to claim 23, 24 or 25 wherein a ' is asparagine.

27. The peptide according to any of claims 23 to 26, wherein a is tyrosine.

28. The peptide according to any of claims 23 to 27, wherein c, a, g' and/! are histidine

29. The peptide according to any of claims 23 to 28, wherein the peptide comprises the sequence:

YGHLEQKHAFLEQKNAALEQHIRALEHK.

30. The peptide according to any of claims 23 to 29, wherein the peptide comprises the following sequence: KYGHLEQKHAFLEQKNAALEQHIRALEHKIRKLE.

31. Use of the peptide according to any one of the preceding claims for determining the presence or absence of an agent.

32. A method of detecting an agent comprising incubating a peptide according to any one of claims 1 to 30 with a test solution, and determining if the peptide has the first structural form or the second structural form in the test solution, wherein if the peptide assumes the second structural form, the test solution contains the agent.