WO2025199069A1 - Rippled beta-sheets and related materials and methods - Google Patents

Abstract

Provided are rippled β-sheets comprising (L)-(MVGGVV)_k (SEQ ID NO:1) dimerized with (D)-(mvggvv)_k (SEQ ID NO:2), (L)-(KLVFFA)_k (SEQ ID NO:3) dimerized with (D)-(klvffa)_k (SEQ ID NO:4), or (L)-(AILSS)_k (SEQ ID NO:5) dimerized with (D)-(ailss)_k (SEQ ID NO:6). Also provided are materials comprising the rippled β-sheets, compositions comprising the materials, and methods of making the rippled β-sheets and materials.

Description

RIPPLED BETA-SHEETS AND RELATED MATERIALS AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/566,811 , filed March 18, 2024, which application is incorporated herein by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (UCSC-410WO_SEQLIST.xml; Size: 22,938 bytes; and Date of Creation: March 17, 2025) is herein incorporated by reference in its entirety.

STATEMENT OF GOVERN ENT SUPPORT

This invention was made with Government support under AG074954, awarded by the National Institutes of Health. The Government has certain rights in the invention.

INTRODUCTION

The incorporation of D-amino acids into proteins and peptides is a versatile strategy that generates mixed chirality frameworks with unique structural and functional properties. Such frameworks have been employed to enhance protein activity,^{1 4} modulate peptide aggregation,^{5 8} and produce peptidic biomaterials with distinctive properties.^9-14 The development of new peptidic systems that contain both L- and D-amino acids has the potential to allow systematic access to an unexplored structural space.

An example of a mixed chirality framework is the rippled p-sheet, first proposed as a theoretical concept by Pauling and Corey in 1953.¹⁵ In a rippled p-sheet D and L peptide strands are arrayed in strictly alternating fashion, giving rise to a distinct backbone topography. Although the rippled p-sheet was conceptualized 70 years ago, a critical mass of experiments in support of its existence has only recently emerged.⁵’ ^9-13’ ^16-24 The history of the field up to 2021 was recently reviewed.²⁵

Previous work demonstrated that racemic mixtures of 40- and 42-residue amyloid-p peptides (D,L-Ap40 and D,L-Ap42) form fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (L-AP40 and L-AP42).^{5 21} This effect, referred to as Chiral Inactivation, protects neuronal model systems from Ap neurotoxicity. Further studies provided insight into the structural basis for Chiral Inactivation.²⁴ It was shown that, rather than the parallel P-sheets formed in naturally observed L-Ap fibrils, the racemic counterparts form antiparallel p- sheets in which D- and L-Ap molecules alternate along the direction of fibril growth, forming a rippled p-sheet. The first atomic resolution structures from a series of model tripeptide systems were subsequently obtained, providing unambiguous structural evidence for the rippled p-sheet.^22, 23

SUMMARY

Provided are rippled p-sheets comprising (L)-(MVGGVV)_k (SEQ ID NO:1 ) dimerized with (D)-(mvggvv)_k (SEQ ID NO:2), (L)-(KLVFFA)_k (SEQ ID NO:3) dimerized with (D)-(klvffa)_k (SEQ ID NO:4), or (L)-(AILSS)_k (SEQ ID NO:5) dimerized with (D)-(ailss)_k (SEQ ID NO:6). Also provided are materials comprising the rippled p-sheets, compositions comprising the materials, and methods of making the rippled p-sheets and materials.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1 D: X-ray crystal structure of the rippled p-sheet formed by MVGGVV (SEQ ID NO:9) hexapeptide and its enantiomer mvggvv (SEQ ID NQ:10), MVGGVV:mvggvv in a mixture of hexaisofluoropropanol (HFIP) and water. (A) A view down the sheet hydrogen-bonding direction reveals rippling of the sheet. L-peptides are shown in green; D-peptides are shown in purple. (B); A view of the sheet face revealing the antiparallel in-register alignment of strands. Backbone H-bond distances (H— O) are reported in angstrom units [A], All backbone amides are involved in stabilizing the rippled sheet except for the amide linkage between G37 and G38, which is rotated out of the plane of the rippled sheet. (C) Crystal packing viewed along the H-bonding direction reveals gaps between sheet faces; contacts are sparse. The unit cell is outlined in black. (D) The gaps between sheet faces are filled by HFIP and water molecules (orange color). The integral role of solvent in maintaining the sheet interface explains why evaporation of HFIP causes crystal dissolution within seconds.

FIG. 2A-2D: X-ray crystal structure of the rippled p-sheet formed by MVGGVV (SEQ ID NO:9) hexapeptide and its enantiomer mvggvv (SEQ ID NQ:10), MVGGVV:mvggvv in a mixture of pentafluoropropionic acid (PFPA) and water. (A) A view down the sheet hydrogen-bonding direction reveals rippling of the sheet. L-peptides are shown in green; D-peptides are shown in purple. (B) A view of the sheet face revealing the antiparallel in-register alignment of strands. Backbone H-bond distances (H— O) are reported in angstrom units [A], All backbone amides are involved in stabilizing the rippled sheet except for the amide linkage between G37 and G38, which is rotated out of the plane of the rippled sheet. (C) Crystal packing viewed along the H-bonding direction reveals the rippled sheets pack face-to-edge rather than face-to-face. The unit cell is outlined. (D) Small gaps in packing are filled by PFPA and water molecules (orange color).

FIG. 3A-3D: X-ray crystal structure of the rippled p-sheet formed by KLVFFA (SEQ ID NO:11 ) hexapeptide and its enantiomer klvffa (SEQ ID NO:12), KLVFFA:klvffa in water and trifluoroacetate (TFA). (A) A view down the sheet hydrogen-bonding direction reveals rippling of the sheet. L-peptides are shown in green; D-peptides are shown in purple. (B) A view of the sheet face revealing the antiparallel out-of-register alignment of strands. Lack of register is indicated by the sideways shift observed with each successive strand up and down the sheet. Backbone Flbond distances (H— O) are reported in angstrom units [A], All backbone amides are involved in stabilizing the rippled sheet. (C) Crystal packing viewed along the H-bonding direction reveals face-to-face packing via the hydrophobic sidechains of L17, V18, F19, and F20. The unit cell is outlined. (D) Small gaps in packing are filled by TFA and water molecules (orange color).

FIG. 4A-4C: X-ray crystal structure of the rippled [3-sheet formed by KLVFFAE (SEQ ID NO:15) hexapeptide and its enantiomer klvffae (SEQ ID NO:16), KLVFFAE:klvffae in water and HFIP. (A) A view down the sheet hydrogen-bonding direction reveals rippling of the sheet. L- peptides are shown in green; D-peptides are shown in purple. (B) A view of the sheet face revealing the antiparallel out-of-register alignment of strands. Lack of register is indicated by the sideways shift observed with each successive strand up and down the sheet. Backbone H-bond distances (H— O) are reported in angstrom units [A]. All backbone amides are involved in stabilizing the rippled sheet. (C) Crystal packing viewed along the H-bonding direction reveals face-to-face packing via the hydrophobic sidechains of L17, V18, F19, F20, and A21 . The unit cell is outlined.

FIG. 5A-5D: X-ray crystal structure of the rippled [3-sheet formed by AILSS (SEQ ID NO:13) pentapeptide and its enantiomer ailss (SEQ ID NO:14), AILSS:ailss in water and HFIP. (A) A view down the sheet hydrogen-bonding direction reveals rippling of the sheet. L-peptides are shown in green; D-peptides are shown in purple. (B) A view of the sheet face revealing the antiparallel in-register alignment of strands. Backbone H-bond distances (H— O) are reported in angstrom units [A], All backbone amides are involved in stabilizing the rippled sheet. (C) Crystal packing viewed along the H-bonding direction reveals face-to-face packing via the hydrophobic sidechains of I26 and L27. The unit cell is outlined. (D) Small gaps in packing are filled by water molecules (orange color).

FIG. 6A-6D: Pleated and Rippled sheets exhibit distinct spatial arrangements of side chains. Sequences in C,D were added geometrically for illustrative purposes. (A) Pleated sheets characteristically arrange side chains (represented as spheres) 4.8 A apart along the hydrogenbonding direction (vertical yellow arrow) and 6.8 A apart along the strand direction (horizontal yellow arrow). (B) Rippled sheets exhibit a wider, more isotropically spaced arrangement of side chains ranging from 5.4 to 6.8 A separation. (C) The narrow spacing (4.8 A) of side chains in pleated sheets creates the opportunity of forming stabilizing ladders of aliphatic, aromatic, or polar residues (esp. Asn and Gin), because side chains are close enough to form favorable van der Waals contacts or hydrogen bonds. On the other hand, the narrow spacing creates repulsion when neighboring side chains carry the same charge. (D) The wider, more isotropic spacing of rippled sheets eliminates the possibility of forming stabilizing ladders but relieves the repulsion when neighboring side chains carry the same charge.

FIG. 7: Comparison of crystallographic p-sheet interfaces of pleated versus rippled geometry. The strongest pleated sheet-sheet interfaces exhibit large areas of surface buried (right side of the graph) and fit together with high complementarity to exclude water (top side of the graph). Six benchmark steric zippers, known to drive (L)-proteins into amyloid fibrils, occupy the upper right corner of the graph. The zippers are viewed down the “fibril” axis. The interface between sheets is highlighted in black. The tightest zippers are those which show the least amount of black in their interface. Rippled sheet interfaces of (L,D)-AILSS (SEQ ID NO:13 and SEQ ID NO:14, respectively) and (L,D)-KLVFFA (SEQ ID NO:11 and SEQ ID NO:12, respectively) are near the steric zipper benchmark, having slightly lower buried surface area but good shape complementarity. Crystal structures of enantiopure (L)-AILSST (SEQ ID NO:17) and (L)-KLVFFA (SEQ ID NO:11 ) pleated sheets are shown for comparison to the corresponding rippled sheet interfaces. The pleated sheet interfaces for these sequences are also outside the benchmark region. This result suggests that rippled sheet interfaces fall outside the benchmark region due to low self-complementarity of the sequences rather than an inherent deficiency of rippled sheets to mate tightly.

FIG. 8: Comparison of -sheet interfaces of rippled geometry (crystallographic) with those of pleated geometry found in amyloid-p fibrils extracted from an Alzheimer’s disease patient (PDB ID 7q4b). The strongest steric zippers exhibit large areas of surface buried (ABu) in the sheetsheet interface and their sheets fit together with high complementarity (Sc) to exclude water (top right corner). Abu and Sc values for 104 pleated sheet interfaces, each 6-residues long, are plotted with gray dots. Two unique zippers from patient-extracted fibrils fall in the “steric zipper benchmark region”. The zippers are viewed down the “fibril” axis. The interface between sheets is highlighted in black. Crystal structures of (L,D)-AILSS (SEQ ID NO:13 and SEQ ID NO:14, respectively) and (L,D)-KLVFFA (SEQ ID NO:11 and SEQ ID NO:12, respectively) rippled sheets are shown for comparison to the pleated interfaces. The rippled sheet interfaces (green/magenta squares) fall outside the benchmark region, but most fall within the region of the segments found in patient-extracted fibrils.

DETAILED DESCRIPTION

Before the rippled 3-sheets, materials and methods of the present disclosure are described in greater detail, it is to be understood that the rippled 3-sheets, materials and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the rippled 3-sheets, materials and methods will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the rippled 3-sheets, materials and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the rippled 3-sheets, materials and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the rippled 3-sheets, materials and methods.

Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the rippled 3-sheets, materials and methods belong. Although any rippled 3-sheets, materials and methods similar or equivalent to those described herein can also be used in the practice or testing of the rippled 3- sheets, materials and methods, representative illustrative rippled 3-sheets, materials and methods are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present rippled p-sheets, materials and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the rippled p-sheets, materials and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the rippled p-sheets, materials and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all subcombinations listed in the embodiments describing such variables are also specifically embraced by the present rippled p-sheets, materials and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

RIPPLED [3-SHEETS AND RELATED MATERIALS AND COMPOSITIONS

Aspects of the present disclosure include rippled p-sheets. The rippled p-sheets comprise a polymer of two or more L-amino acids dimerized with a polymer of two or more D-amino acids. The term “amino acid” generally refers to any monomer unit that comprises a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox- active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties.

The term “amino acid” includes, but is not limited to, naturally-occurring a-amino acids and their stereoisomers. “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally- occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid (/.e., the D-amino acid). “L” refers to levorotatory and “D” refers to dextrorotatory.

Naturally-occurring a-amino acids are those encoded by the genetic code as well as those amino acids that are later modified (e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine). Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (lie), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally-occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D- lle), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-GIn), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. For example, an L-amino acid may be represented herein by its commonly known three letter symbol (e.g., Met for L-methionine) or by an upper-case one-letter amino acid symbol (e.g., M for L-methionine). A D-amino acid may be represented herein by its commonly known three letter symbol (e.g., D-Met for D-methionine) or by a lower-case one-letter amino acid symbol (e.g., m for D-methionine).

The terms “polypeptide,” “peptide,” “protein”, and “polymer of amino acids,” used interchangeably herein, refer to a polymeric form of amino acids of any length (e.g., connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues), which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

In certain embodiments, the rippled p-sheets comprise (L)-(MVGGVV)_k (SEQ ID NO:1 ) dimerized with (D)-(mvggvv)_k (SEQ ID NO:2); (L)-(KLVFFA)_k (SEQ ID NO:3) dimerized with (D)- (klvffa)_k(SEQ ID NO:4); or (L)-(AILSS)_k (SEQ ID NO:5) dimerized with (D)-(ailss)_k (SEQ ID NO:6), where k is an integer of 1 or greater. In some instances, such a rippled p-sheet comprises (L)- (KLVFFAE)_k (SEQ ID NO:7) dimerized with (D)-(klvffae)_k (SEQ ID NO:8).

According to some embodiments, k is an integer of from 1 to 1000, such as from 1 to 750, from 1 to 500, from 1 to 250, from 1 to 100, from 1 to 75, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, or from 1 to 10.

In certain embodiments, the rippled p-sheets are water-soluble, i.e., capable of being dissolved in water. According to some embodiments, the termini of the monomers of the dimers comprise a moiety that renders the dimers water-soluble. Examples of such moieties include, but are not limited to, a free amine, a free carboxylate, and/or the like.

Aspects of the present disclosure further include materials comprising a plurality of the rippled p-sheets of the present disclosure.

Also provided are compositions comprising the materials of the present disclosure. In certain embodiments, such a composition includes a material of the present disclosure present in a liquid medium, e.g., an aqueous liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCL, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine- N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

A surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1 % w/v.

A lyoprotectant may also be added in order to protect the materials against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

In certain embodiments, a composition of the present disclosure comprises the material and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, tricresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the composition, e.g., at concentrations ranging from about 0.001 to about 2% weight/volume (w/v). METHODS

Also provided by the present disclosure are methods of making and using the rippled [3- sheets, materials and compositions of the present disclosure.

According to some embodiments, provided are methods comprising producing a polypeptide comprising, consisting essentially of, or consisting of (L)-(MVGGVV)_k(SEQ ID NO:1 ); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)-(mvggvv)_k (SEQ ID NO:2).

In certain embodiments, provided are methods comprising producing a polypeptide comprising, consisting essentially of, or consisting of (L)-(KLVFFA)_k (SEQ ID NO:3); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)-(klvffa)_k (SEQ ID NO:4). According to some such embodiments, the methods comprise producing a polypeptide comprising, consisting essentially of, or consisting of, (L)-(KLVFFAE)_k (SEQ ID NO:7); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D(D)-(klvffae)_k (SEQ ID NO:8).

In some instances, the polypeptides are produced by chemical synthesis. A non-limiting example of a chemical synthesis includes solid-phase synthesis (e.g., solid-phase peptide synthesis). Solid-phase peptide synthesis (SPPS) involves the successive addition of protected amino acid derivatives to a growing peptide chain immobilized on a solid phase, including deprotection and washing steps to remove unreacted groups and also side products. Any number of solid supports may be employed, including resins such as polystyrene and polyamide based resins. The peptides may be covalently bound to a solid support, typically at their C-terminal end through linkers such as acid labile and photolabile linkers. In some embodiments the linker is an acid labile linker. In other embodiments, the linker is a trityl linker such as a 2-chlorotrityl linker. Peptide synthesis is typically performed by coupling a protected amino acid to the N-terminal end of the bound sample. The protected amino acid may contain N-terminal protecting groups such as a Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl) group as well as side chain protecting groups. According to some embodiments, when solid-phase polypeptide synthesis is performed to produce the polypeptides, the solid-phase synthesis is Fmoc-based solid-phase synthesis.

The above-described methods may further comprise purifying the produced polypeptides. Any suitable approach for purifying the polypeptides may be employed. In certain embodiments, the polypeptides are purified by chromatography, a non-limiting example of which is High Performance Liquid Chromatography (HPLC). An example HPLC-based approach suitable for purifying the polypeptides is described in Warner et al. (2017) JoVE, 2017, e55482.

The above-described methods may further comprise combining the produced polypeptides into a racemic mixture. In certain embodiments, the combining is under conditions suitable for formation of the rippled [3-sheets of the present disclosure. For example, according to some embodiments, provided are methods that comprise combining the L- and D-polypeptides in a mixture under conditions in which the rippled [3-sheets are formed. Non-limiting examples of suitable conditions for forming the rippled [3-sheets of the present disclosure are described in detail herein, e.g., in the Experimental section below.

According to some embodiments, the conditions comprise combining the polypeptides in the presence of a fluorinated solvent. Non-limiting examples of fluorinated solvents which may be employed when practicing the methods of the present disclosure include hexafluoroisopropanol (HFIP) and pentafluoropropionic acid (PFPA).

Non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

1 . A rippled [3-sheet comprising:

(L)-(MVGGVV)_k (SEQ ID NO:1 ) dimerized with (D)-(mvggvv)_k(SEQ ID NO:2);

(L)-(KLVFFA)_k (SEQ ID NO:3) dimerized with (D)-(klvffa)_k(SEQ ID NO:4); or (L)-(AILSS)_k (SEQ ID NO:5) dimerized with (D)-(ailss)_k(SEQ ID NO:6), wherein k is an integer of 1 or greater.

2. The rippled [3-sheet of clause 1 , comprising (L)-(KLVFFAE)_k (SEQ ID NO:7) dimerized with (D)-(klvffae)_k(SEQ ID NO:8).

3. A material comprising a plurality of the rippled [3-sheets of clause 1 or 2.

4. A composition comprising the material of clause 3.

5. The composition of clause 4, wherein the material is present in a liquid medium.

6. A method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (MVGGVV)_k (SEQ ID NO:1 ); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (mvggvv)_k (SEQ ID NO:2).

7. A method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (KLVFFA)_k (SEQ ID NO:3); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (klvffa)_k (SEQ ID NO:4).

8. The method of clause 7, comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (KLVFFAE)k(SEQ ID NO:7); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (klvffae)k(SEQ ID NO:8).

9. A method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (AILSS)k (SEQ ID NO:5); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (ailss)k (SEQ ID NO:6).

10. The method of any one of clauses 6-9, wherein the polypeptides are produced by chemical synthesis.

11 . The method of clause 10, wherein the chemical synthesis is by solid-phase synthesis.

12. The method of clause 11 , wherein the solid-phase synthesis is Fmoc-based solid-phase synthesis.

13. The method of any one of clauses 6-12, further comprising purifying the produced polypeptides.

14. The method according to any one of clauses 6-13, further comprising combining the produced polypeptides into a racemic mixture.

15. A method comprising : combining the polypeptides produced according to the method of any one of clauses 6- 13 in a mixture under conditions in which rippled p-sheets are formed.

16. The method of clause 15, wherein the conditions comprise combining the polypeptides in the presence of a fluorinated solvent.

17. The method of clause 16, wherein the fluorinated solvent is hexafluoroisopropanol (HFIP) or pentafluoropropionic acid (PFPA).

The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL

Reported herein are results from atomic-resolution crystallographic studies on racemic mixtures of peptides from segments of the disease forming proteins, A|3 and Amylin. The motivation for pursuing this work was three-fold. Given that only a handful of atomic-resolution structures of rippled-sheets have been obtained and are limited to tripeptide sequences composed of aromatic residues, the first objective was to expand the crystallographic foundation to include sequences of greater length and complexity. Structural evidence for the formation of rippled sheets from more complex racemic peptides such as MAX1 ,⁹' ¹⁰ KFE8,¹¹’ ¹³ Ac-KLVFFAE- NH₂ ¹³ and YTIAALLSPYS (SEQ ID NO:18),²⁶ has only been obtained by indirect, biophysical methods.

Secondly, the results herein in support of rippled sheets in D,L-A[340 fibrils suggest that rippled sheet formation is a generic property of racemic mixtures of amyloid-like peptide assemblies. This hypothesis was tested further by crystallizing short fragments from peptides that form amyloid-like fibrils. Furthermore, the choice of these peptide systems would permit comparisons with solution-state NMR studies²⁷ on model peptides from A[3 which show a clear preference for homochiral assembly over heterochiral assembly.

A third objective was to compare atomic resolution structures of racemic crystals to those of existing enantiopure crystals. Both crystal types assemble on a cross-beta scaffold, where individual beta-strands orient perpendicular to the length of the crystalline needle, and stack in beta-sheets that run the entire length of the needle. Crystals of enantiopure amyloidogenic peptides have typically revealed tight mating between adjacent beta-sheet surfaces, excluding water from their interface. This structural motif, called a “steric-zipper”, lends amyloid fibrils their extraordinary stability.²⁸ It was queried whether rippled sheets are capable of mating in an analogous fashion or whether a different structural space would emerge.

To address these objectives, selected were four distinct peptide segments from A|3 and Amylin, which varied in hydrophobicity, charge, and sequence length. All four sequences formed extended, antiparallel rippled p-sheets. The present findings demonstrate a clear preference for heterochiral rippled p-sheets over homochiral pleated p-sheets upon crystallization and suggest that the tendency for racemic peptides to form rippled p-sheets in three-dimensional systems is much greater than previously thought. Racemic peptides from Amyloid beta and Amylin form rippled B-sheets rather than pleated B- sheets

Previous work identified the hydrophobic, aromatic residues Phe, Tyr and Trp as ripple- genic.^{22, 23} To determine whether sequences composed predominantly of non-aromatic, hydrophobic residues could form rippled sheets, the MVGGVV (SEQ ID NO:9) segment from the C-terminus of AB (A 35-40) was selected. A racemic mixture of MVGGVV (SEQ ID NO:9) and mvggvv (SEQ ID NQ:10) was crystallized from a solution of hexafluoroisopropanol (HFIP) in water, yielding needles of antiparallel rippled B-sheet layers as determined by X-ray crystallography (Figure 1A,B). The rippled interface is partially interrupted at the G37-G38 junction. As observed in the previously reported tripeptide structures, the individual tripeptides stack in the H-bonding dimension, forming extended antiparallel rippled B-sheet layers, in which mirror image peptide strands are arranged in strictly alternating fashion. Each L-hexapeptide (Figure 1 B) is sandwiched between two D-hexapeptides (Figure 1 B), and each D-hexapeptide is sandwiched between two L-hexapeptides in periodic fashion within the individual B-sheets, with H-bond distances ranging from 1 .910(1 ) to 2.050(1 ) A. Each hexapeptide has six H-bonds to one of its two direct neighbors in the layer (i.e., “tight dimer”), and four H-bonds to the other (i.e. , “loose dimer”) (Figure 1 B).

The torsional angles for the glycine residues G37 and G38 are measured as phi -69.04 and i = 151 .8 and, (p = -74.8 and i = 168.6, respectively for the L-hexapeptide, and <p = 69.0 and ip = -151.8 and, <p = 74.8 and ip = -169.6, respectively, for the inversion-related D-hexapeptide. The G37 and G38 (p angles are considerably smaller than those found in crystals of enantiomerically pure MVGGVV (SEQ ID NO:9), resulting in a ~90-degree rotation of the G37- G38 peptide plane from its standard orientation in a B-sheet.²⁸ An additional consequence of this rotation is the positioning of the V36 and V39 sidechains; in the rippled sheet the sidechains reside on the same face of the B-sheet whereas in the pleated sheet V36 resides on the opposite face of the B-sheet from V39.²⁸

Unlike many amyloid-like structures²⁸ and the tripeptide rippled sheet structures reported perviously,²² pairs of rippled antiparallel sheets do not form a tight dry interface; instead, the sheets form a cavity, which encapsulates the HFIP molecules (Figure 1C,D). Pairs of rippled sheets are also associated in the lateral direction through the formation of salt bridges between N and C-termini (Figure 1 C, dashed lines). As a result, an additional cavity, running parallel to the fibril axis, forms between the salt bridges and the side chains of terminal residues of the rippled sheets; HFIP molecules are also trapped in this cavity and are hydrogen bonded to the carboxylate and ammonium termini of the loose dimer. Formation of the three-dimensional crystallographic lattice appears to be contingent on the presence of HFIP molecules, as exposure to air leads to the dissolution of the crystals after several minutes.

Given the presence of a cavity, assessed was whether other fluorinated solvents could be incorporated into the extended crystallographic lattice and whether they would influence the long- range packing. A polymorph of MVGGVV:mvggvv was obtained by crystallization of a racemic mixture from a solution of water and pentafluoropropionic acid (PFPA). The resulting needles contained periodic MVGGVV:mvggvv antiparallel rippled [3-sheet layers, as determined crystallographically (Figure 2 A,B).

The packing of the individual hexapeptides in the H-bonding direction closely resembles that observed in the MVGGVV:mvggvv HFIP structure, alternating between tight and loose dimers. H-bond distances range between 1.969(9) to 2.166(9) A. The torsional angles for the glycine residues G37 and G38 are measured as <p = -68.2 and 153.1 and, <p = -68.8 and ip = 169.0, respectively for the L-peptide and, ) = 68.1 and qj = - 151 .7 and, q> = 68.2 and ip = -169.0, respectively for the D-peptide. As observed in the MVGGVV:mvggvv HFIP structure, the G37- G38 peptide plane is rotated ~90 degrees from its standard orientation in a [3-sheet and the V36 and V39 sidechains reside on the same face of the p-sheet.

While the structures of the rippled dimers of MVGGVV:mvggvv HFIP and PFPA display close resemblance to each other, the three-dimensional crystallographic lattices of the structures differ considerably (Figure 2C,D). In the PFPA structure, pairs of rippled antiparallel sheets do not pack in a face-to-face arrangement but rather, pack face-to-edge. A water molecule connecting G37 of one strand to G38 on the strand of the opposite chirality, in the tight dimer, also interacts with the N-terminus of the dimer in the opposite orientation, connecting face to edge or edge to face dimers. PFPA molecules are trapped in the crystal lattice but are not found between rippled sheets. Instead, the PFPA molecules bind to the N and C termini of the loose dimer. The variation in long-range packing between the MVGGVV:mvggvv polymorphs most likely arises from differing protonation states at the C-terminus; PFPA is strongly acidic and can protonate the C-terminus of the peptide, while HFIP being only mildly acidic, cannot.

Encouraged by the ability to resolve the structures of these rippled beta sheets, the central hydrophobic region of A[3 was explored. The KLVFFA (SEQ ID NO:11) segment (A[316-21 ) contains a charged residue at the N terminus as well as both aromatic and alkyl hydrophobic residues. A racemic mixture of KLVFFA (SEQ ID NO:1 1 ) and klvffa (SEQ ID NO:12) was crystallized from a solution of HFIP in water, yielding needles of antiparallel rippled -sheets. The individual hexapeptides stack in the H-bonding dimension, forming extended antiparallel rippled p-sheet layers (Figure 3A,B), similar to those observed in the structure of MVGGVV:mvggvv. In contrast to MVGGVV:mvggvv, the hexapeptide strands are tilted with respect to the “fibril” axis, resulting in an out-of-register sheet. As a consequence, only tight dimers are found in the KLVFFA:klvffa structure; each hexapeptide forms six hydrogen bonds to its two neighbors in the rippled sheet, with H-bond distances ranging from 1 .92(2) to 2.07(3) A. In addition to the hydrogen bonds, CH-pi interactions are observed between the methylene protons of the K16 sidechain and the aromatic ring of F20 on the strand of opposite chirality.

Tight packing between the hydrophobic side chains of L17, V18, F19 and F20 results in the formation of a dry interface between pairs of sheets (Figure 3C,D). CH-pi interactions are observed at the interface between the side chains of L17 and the aromatic rings of F19 and F20. In the lateral direction, the rippled dimers interact via salt bridges; the carboxylate terminus forms a salt bridge to the ammonium group of the K16 sidechain as well as to the amino group of the N terminus of the neighboring dimer.

Examined next was the effect of adding an additional charge to the peptide sequence by expanding it C-terminally. The KLVFFAE (SEQ ID NO:15) segment of A|3 (A[3 16-22) contains polar charged residues at both the N and C termini. Crystals of Ac-KLVFFAE-NH₂ and Ac-klvffae- NH₂were grown from a solution of HFIP in water. X-ray and electron diffraction from the resulting needles extended to approximately 2.0 A resolution, and a chemically reasonable model could be obtained by molecular replacement using an ideal p-strand as a search model. Difference density revealed the positions of the side chains. However, this atomic model produced unsatisfactory refinement statistics (R-work 0.465/R-free 0.490). Crystal disorder was attributed as the cause of the discrepancy between the atomic model and the diffraction data. The specific type of disorder is unclear since twinning and translocation corrections produced no improvement in R-factors. Despite the limitations of the model, several key features of the structure could be elucidated (Figure 4A,B). The individual heptapeptides stack in the H-bonding dimension, forming extended antiparallel rippled p-sheet layers, with alternation of the L and D peptides. As observed with KLVFFA:klvffa, the heptapeptide strands are tilted with respect to the unit cell, forming an out of register sheet, with tight dimers between neighboring strands; H-bond distances range from 1.92(10) to 2.81 (11 ) A. Interestingly, the strand registration differs from KLVFFA:klvffa. In the KLVFFAE:klvffae rippled dimer, F19 stacks with F19 on the adjacent strand whereas in KLVFFA:klvffa, V18 stacks with V18 on the adjacent strand.

Additionally, in the KLVFFAE :klvffae structure, the strands from adjacent sheets cross with an "X" pattern (Figure 4C) whereas in the KLVFFA:klvffa structure, all the strands run parallel/antiparallel to the same, single axis. A study by Nilsson on the self-assembly of I- and d- KLVFFAE (SEQ ID NO:15 and SEQ ID NO:16, respectively) by FTIR and solid-state NMR proposes an in-register structure in which the antiparallel rippled sheets are precisely aligned.¹³ The structural model supports the proposed rippled antiparallel dimer but differs in that the individual dimers are offset with respect to each other within the sheet.

To explore whether the rippled sheet architecture is sufficiently robust to support sequences that include small polar residues, the pentapeptide sequence AILSS (SEQ ID NO:13) from amylin (residues 25-29) was selected for crystallization. A racemic mixture of AILSS (SEQ ID NO:13) and ailss (SEQ ID NO:14) was crystallized from HFIP and water. Analysis of the needles by X-ray crystallography indicated the formation of antiparallel rippled sheets. Each pentapeptide strand forms 4-H bonds to each of its neighbors with H-bond distances between the rippled dimers ranging from 2.018(11 ) to 2.123(10) A (Figure 5A,B). The phi torsional angles for the two Ser residues differ considerably and are measured as <p = -145.03 for S28 and q> = -77.53 for S29. The S29 residue presumably adopts this conformation to hydrogen bond with the N- terminus of the adjacent strand. Rippled sheets mate face-to-face via a dry interface formed by packing of the hydrophobic side chains of I26 and Leu27 (Figure 5C,D). In contrast, the serine residues are surrounded by water molecules. Two water molecules connect S28 to the C terminus of a strand across the steric zipper.

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Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1. A rippled p-sheet comprising:

(L)-(MVGGVV)_k (SEQ ID NO:1 ) dimerized with (D)-(mvggvv)_k(SEQ ID NO:2);

(L)-(KLVFFA)_k (SEQ ID NO:3) dimerized with (D)-(klvffa)_k(SEQ ID NO:4); or (L)-(AILSS)_k (SEQ ID NO:5) dimerized with (D)-(ailss)_k(SEQ ID NO:6), wherein k is an integer of 1 or greater.

2. The rippled [3-sheet of claim 1 , comprising (L)-(KLVFFAE)_k (SEQ ID NO:7) dimerized with (D)-(klvffae)_k(SEQ ID NO:8).

3. A material comprising a plurality of the rippled p-sheets of claim 1 .

4. A composition comprising the material of claim 3.

5. The composition of claim 4, wherein the material is present in a liquid medium.

6. A method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (MVGGVV)_k (SEQ ID NO:1 ); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (mvggvv)_k (SEQ ID NO:2).

7. A method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (KLVFFA)_k (SEQ ID NO:3); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (klvffa)_k (SEQ ID NO:4).

8. The method of claim 7, comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (KLVFFAE)_k(SEQ ID NO:7); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (klvffae)_k(SEQ ID NO:8).

9. A method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of (L)- (AILSS)k (SEQ ID NO:5); and producing a polypeptide comprising, consisting essentially of, or consisting of, (D)- (ailss)k (SEQ ID NO:6).

10. The method of any one of claims 6-9, wherein the polypeptides are produced by chemical synthesis.

11 . The method of claim 10, wherein the chemical synthesis is by solid-phase synthesis.

12. The method of claim 1 1 , wherein the solid-phase synthesis is Fmoc-based solid-phase synthesis.

13. The method of any one of claims 6-9, further comprising purifying the produced polypeptides.

14. The method according to any one of claims 6-9, further comprising combining the produced polypeptides into a racemic mixture.

15. A method comprising : combining the polypeptides produced according to the method of any one of claims 6-9 in a mixture under conditions in which rippled p-sheets are formed.

16. The method of claim 15, wherein the conditions comprise combining the polypeptides in the presence of a fluorinated solvent.

17. The method of claim 16, wherein the fluorinated solvent is hexafluoroisopropanol (HFIP) or pentafluoropropionic acid (PFPA).