EP4669336A1

EP4669336A1 - DESIGN OF AMYLOIDOGENIC PEPTIDE TRAP

Info

Publication number: EP4669336A1
Application number: EP24741858.5A
Authority: EP
Inventors: David Baker; Danny SAHTOE; Hannah Han
Original assignee: University of Washington
Current assignee: University of Washington
Priority date: 2023-01-11
Filing date: 2024-01-09
Publication date: 2025-12-31
Also published as: CN120548189A; KR20250133678A; WO2024151580A1

Abstract

Polypeptides having an amino acid sequence at least 50% identical, not including any insertions, to the amino acid sequence of one of SEQ ID NO: 1-18, 31-36, and 43-45 are provided and their use, where the polypeptides include a plurality of alpha helical and beta, strand domains, and bind to a target peptide capable of forming a P-strand.

Description

Design of amyloidogenic peptide traps Cross Reference This application claims priority to U.S. Provisional Application Serial Number 63/479,354 filed January 11, 2023, incorporated by reference herein in its entirety. Sequence Listing Statement A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on January 7, 2024 having the file name “22- 2196-WO.xml” and is 45,340 bytes in size. Background Many proteins contain segments that only become ordered upon binding a target. A particularly interesting example of such disorder-to-order transitions are amyloidogenic sequences found in proteins such as Aȕ42, Tau and Serum amyloid A1. These regions can aggregate into amyloid fibrils via strand-strand interactions and are associated with amyloidosis and associated diseases both inside and outside the central nervous system. Designed binders to amyloid forming segments of these proteins could have utility both as diagnostics and therapeutics. However, it is difficult to raise antibodies against the monomeric form of amyloid due to their strong tendency for self-association; this also complicates the systematic generation of binders using library selection methods. The multiplicity of conformations complicates design of binders to disordered protein segments, and the computational design of binders to amyloid forming segments of proteins remains an outstanding challenge. Summary In one aspect, the disclosure comprises polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:1-18, 31-36, and 43-45, wherein the polypeptide includes a plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a ȕ-strand. In one embodiment, the polypeptide comprises at least 4 alpha helices and at least 4 beta strands. In another embodiment, any substitution of binding pocket interface residues is a conservative amino acid substitution relative to the reference sequence. In a further embodiment, binding pocket interface residues are conserved relative to the reference sequence. In another embodiment, amino acid residues in loop regions may be substituted with any other amino acid; optionally wherein amino acid residues in loop regions may be substituted with any other amino acid other than proline. In one embodiment, any substitutions relative to the reference sequence at amino acid residues in beta strand or alpha helical domains are conservative amino acid substitutions. In a further embodiment, any substitutions relative to the reference sequence are conservative amino acid substitutions. In another embodiment, the polypeptides comprise an insertion in a loop region, at the amino-terminus or the polypeptide, and/or at the C-terminus of the polypeptide. In another aspect, the disclosure provides kits comprising a combination of polypeptides, selected from the group consisting of: (a) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1; and (ii) a second polypeptide comprising or consisting of the amino acid sequence Gqrirvritg (SEQ ID NO:19); (b) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:2; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqricvritg (SEQ ID NO:20); (c) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1 or 4; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqrirvcitg (SEQ ID NO:21); (d) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1 or 4; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqrirvritg (SEQ ID NO:19); (e) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:5; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvtfivhe (SEQ ID NO:23); (f) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:6; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfqvre (SEQ ID NO:24); (g) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:7; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfnfre (SEQ ID NO:25); (h) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:8; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfnfre (SEQ ID NO:25); (i) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:9; and (ii) a second polypeptide comprising or consisting of the amino acid sequence sqthfevefkgmrirlrns (SEQ ID NO:27); (j) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:10; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gwlefeyekngrvirlvqg (SEQ ID NO:28); (k) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:11; and (ii) a second polypeptide comprising or consisting of the amino acid sequence sqtqfeyekngrrirlrqs (SEQ ID NO:29); and/or (l) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:12; and (ii) a second polypeptide comprising or consisting of the amino acid sequence ssvrveehmngvriqmeyg (SEQ ID NO:30). In various other aspects, the disclosure provides nucleic acids encoding the polypeptide of any embodiment of the disclosure; expression vectors comprising a nucleic acid of any embodiment of the disclosure operatively linked to a suitable control element; and recombinant host cells comprising the polypeptide, kit, nucleic acid, and/or expression vector of any embodiment of the disclosure. In another aspect, the disclosure provides methods for treating an amyloid disease or amyloid-associated disease, comprising administering to a subject in need thereof an amount effective to treat the condition of the polypeptide of any embodiment of the disclosure. In a further aspect, the disclosure provides methods for diagnosing, prognosing, or monitoring an amyloid disease or amyloid-associated disease, comprising (a) contacting a tissue sample from a subject at risk of having an amyloid disease or amyloid-associated disease with the polypeptide of any embodiment of the disclosure, under conditions suitable for binding of the polypeptide with amyloid, if present in the tissue sample, to produce a binding complex (b) detecting binding complexes in the tissue samples; and (c) diagnosing or prognosing an amyloid disease or amyloid-associated disease based on the detecting. Description of the Figures Figure 1. Design approach for binding disordered protein fragments. (a) Intrinsically disordered regions of proteins and peptides have large conformational freedom but may be forced into predefined conformations such as ȕ-strands that can be efficiently targeted using strand-strand interactions. (b) Molecular mechanics simulation of a model peptide shows it adopts a wide range of conformations (left) but can be modeled in a ȕ- conformation while strand-pairing to a de novo protein (middle). A second domain (right) can be designed that provides strand-strand interactions to the other side of the peptide creating a single chain protein with a deep and complementary peptide binding cleft (SEQ ID NO: 19). Figure 2. Characterization of designed peptide binders. Designed models for peptide binders (binder in lighter shade, peptide in darker shade) (Top: from left to right: SEQ ID NOs: 24, 25 and 19; Bottom: from left to right: SEQ ID NOs: 27, 29 and 30). Respective SEC (S75 increase 10/300) chromatograms of the binders are shown below the models. Figure 3. Interfaces and affinity maturation. (a) Detailed views of the solvent exposed interface of C37 (bottom) and the buried interface (top). C-alpha atoms as spheres. (b) Detailed view of the buried part of the interface of hairpin binder CH17. (c) Models of parent design C34 (top) and C34.1 (bottom) where an hydrophobic interaction pair (sticks and spheres) is introduced to improve affinity. Figure 4. Design of disulfide stabilized complexes. (a) View of the designed interface disulfide on C104.3 (disulfide in spheres and sticks; additional redesigned residues in sticks). (b) Non reducing SDS-PAGE gel showing disulfide formation (timepoints; t=0, t=90min t=overnight). (c) SEC trace of preformed non-covalent C104 complex + GFP- pep104. (d) SEC trace of preformed covalent disulfide linked C104.3 complex + GFP- pep104. Figure 5. Structural characterization of designs. On the left; Overlay of the design model of a surface redesigned version of C104 and the crystal structure (On the right; Detailed interface view of design and crystal structure with Ile8 shift indicated with dotted arrow. Figure 6. Design of amyloid peptide traps. Amyloidogenic proteins can assemble into fibrils (top). To modulate fibril formation we design binders (middle) that can sequester a region (dark) that participates in fibril formation in its binding cleft to inhibit fibril formation. Figure 7. Characterization of amyloid peptide traps. BLI binding experiments of designed proteins binding to their corresponding amyloidogenic target peptide. From left to right and top to bottom: SEQ ID NOs: 22, 26, 37, 39, 38, and 40). Fig 8. C104 controls. (a) SEC binding assay showing that a fusion protein between GFP and 104 peptide binds to the C104 design on a S75 increase 10/300. (b) Close-up view of the buried part of the C104 interface with Val6 shown in sticks and spheres. (c) Biolayer interferometry trace of C104 binding to base peptide 104 and to a peptide with a V6R substitution. (d) Interface close up view of C104 highlighting the hydrophobic-hydrophilic pattern of the peptide. Buried residues single letter amino acid identifiers are underlined. Fig 9. Computational affinity maturation by introducing solvent exposed hydrophobic interaction pairs. (a) View of the solvent exposed interface of CH15. (b) View of the redesigned CH15.1 interface. Hydrophobic interaction pairs introduced to the base CH15 scaffold to improve affinity are highlighted in sticks and spheres. Superdex^TM 75 Increase 10/300 GL SEC traces of purified C34.1 (c) and CH15.1 (d). Fig 10. Disulfide functionalization of C104. (a) Close-up of C104 surface exposed interface (top) and of the disulfide bridge variants C104.2 (middle) and C104.3 (bottom). Disulfide bonds are highlighted with spheres while additional redesigned residues to optimally accommodate the disulfide bridges are highlighted in thicker sticks. (b) Coommassie stained non-reducing SDS-PAGE gel monitoring disulfide bridge formation of C104.2. Time points are t=0, t=90min and t=overnight. Fig 11. Incorporation of C37 into LHD hetero-oligomer system. Design C37 was rigidly fused to LHD284B_DHR9 (right) creating a single chain protein with two interfaces capable of binding the peptide of C37 and the designed binding partner of LHD284B_DHR9, LHD284A_DHR82. We validated the assembly of this ternary complex in a SEC binding assay on a S200 increase 10/300 GL. A: GFP-peptC37, B: GFP-peptC37 + LHD284B_DHR9, C: GFP-peptC37 + LHD284B_DHR9 + LHD284A_DHR82. Absorbance at 395 nm of the GFP-peptC37 was monitored to assess binding. Fig 12. Amyloidogenic sequence docking. The designed peptides are optimized to only harbor GFAMILYVWST (SEQ ID NO:42) residues at the buried positions; charged residues cannot be accommodated at buried positions due to the high chance of burying a polar residue that cannot be satisfied by complementary side chains on the scaffold. To identify stretches of sequence present in amyloidogenic proteins that can be accommodated in a beta strand conformation in the binding pockets of the designs (fig 2a), the burial pattern of the peptides (middle) are matched to the primary sequence of the amyloidogenic protein (top). Because prolines disrupt beta conformation they are only allowed at termini. When a match is found the original peptide sequence is mutated to the matched sequence from the amyloid protein (threading) and docked back into the scaffold binding pocket followed by redesign of the scaffold interface residues (sticks) to optimize interactions to the amyloidogenic sequence (right). Detailed Description All references cited are herein incorporated by reference in their entirety. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In all embodiments of polypeptides disclosed herein, any N-terminal methionine residues are optional (i.e.: the N-terminal methionine residue may be present or may be deleted, and if deleted the residue is not considered when determining percent identity). All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise. Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:1-18, 31-36, and 43-45, wherein the polypeptide includes plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a ȕ-strand . As described in the examples, segments of proteins with ȕ-strand propensity can self associate to form amyloid fibrils associated with many diseases. These regions often adopt alternative structures in their folded states, or are intrinsically disordered in solution, making it difficult to generate binders or inhibitors with existing strategies. The inventors describe a general approach to bind such segments in ȕ-strand and ȕ-hairpin conformations using de novo designed scaffolds, exemplified by the polypeptides of the disclosure, that contain deep peptide binding clefts flanked by ȕ-strands that form hydrogen bonds to the peptide upon binding. The amino acid sequence of SEQ ID NO:1-18, 31-36, and 43-45 are provided in Table 1; immediately below each sequence is an annotation showing the secondary structure of each polypeptide where “E” identifies residue as being present in a beta strand (with a contiguous stretch of 3 or more “E” residues identifying a beta strand), “H” identifies a residue as being present in an alpha helix (strand (with a contiguous stretch of 4 or more “H” residues identifying an alpha helix), and “L” identifies a residue as being present in a loop region between alpha helices and/or beta strands. Within the amino acid sequences, lower case font identifies the residue as being a binding pocket interface residue. Table 1 The polypeptides comprise a plurality (i.e., at last two) of alpha helical and beta strand domains and bind to a target peptide capable of forming a ȕ-strand. The polypeptides contain deep peptide binding clefts flanked by ȕ-strands that form hydrogen bonds to the peptide upon binding. The polypeptides may have any number of alpha helices and beta strands. In one non-limiting embodiment, the polypeptides comprise at least 4 alpha helices and at least 4 beta strands. In some embodiments, any substitution relative to the reference polypeptide sequence of binding pocket interface residues (i.e., residues in lower case in the sequences shown in Table 1) is a conservative amino acid substitution. In other embodiments, binding pocket interface residues are conserved (i.e., identical) relative to the reference sequence. By way of example, the amino acid sequence and secondary structure of SEQ ID NO:31 are provided below. HLTAU_014: Target amyloid forming protein: Tau Sequence spgvvlgefervDdaLEaaERlAEEaQKLLDEYAAQLRSLAEEYIKQKEPYGQQILNELEEF EEAKENRAKIYVEDdvvyvehPLlDAITNSLDILEEHRPELAYLKEEFEEALAATlQEThDR YVAEFEK (SEQ ID NO:31) Secondary structure LLLEEEEEELLHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHLLLHHHHHHHHHHHHH HLLLLLLLEEEEELLEEEEELHHHHHHHHHHHHHHHHLHHHHHHHHHHHHHHHHHHHHHHHH HHHLLLL Binding pocket interface residues are shown in lower case and bold font. Thus, the following residues in SEQ ID NO:31 are binding pocket interface residues (i.e., at the interface with the target amyloid): 1-12, 14-15, 18-19, 22, 26, 77-83, 86, 118, and 122. The secondary structure element in which each residue is present in SEQ ID NO:31 is provided in the “secondary structure” notation (H + helix; L= loop and underlined, E = beta strand and bold font). Thus, the positions of amino acid residues in the secondary structure of SEQ ID NO:31 is as follows: Loop 1: Residues 1-3; Beta strand 1: Residues 4-9; Loop 2: Residues 10-11; Helix 1: Residues 12-46; Loop 3: Residues 47-49; Helix 2: Residues 50-63; Loop 4: Residues 64-70; Beta strand 2: Residues 71-75; Loop 5: Residues 76-77; Beta strand 3: Residues 78-82; Loop 6: Residue 83; Helix 2: Residues 84-99; Loop 7: Residue 100; Helix 3: Residues 101-127; and Loop 8: Residues 128-131. Those of skill in the art will understand how to identify binding pocket interface residue positions and residues present in different structural elements based on the information disclosed herein. As described in the examples, mutagenesis studies on HLTAU014 (SEQ ID NO:31) were carried out, and showed that at least the following binding pocket interface residues could be modified, as shown in Table 2. These studies also identified improved HLTAU014 variants, listed in Table 1 as SEQ ID NO:35 and 36. Thus, in some embodiments, the polypeptides may comprise 1, 2, 3, 4, or all 5 substitutions compared to SEQ ID NO:31, 35, or 36, selected from the group consisting of: (a) V4I or V4Y; (b) L6V; (c) R11T or T11R; (d) H83D, H83N, or N83H; and (e) H122L. Table 2 As described in the examples, mutagenesis studies on 2HLTAU_011 (SEQ ID NO:33) were carried out, and showed that at least the following binding pocket interface residues could be modified, as shown in Table 3. Thus, in some embodiments, the polypeptides may comprise 1, 2, 3, 4, 5, 6 or all 7 substitutions compared to SEQ ID NO:33, selected from the group consisting of: (a) V6Y or V6W; (b) I11M; (c) A41Y; (d) Q44E; (e) P80R or P80A; (f) R106N or R106Q; and (g) M107W. Table 3 As described in the examples, mutagenesis studies on DAM_015 (SEQ ID NO:16) were carried out, and showed that at least the following binding pocket interface residues could be modified, as shown in Table 4. Thus, in some embodiments, the polypeptides may comprise 1, 2, 3, 4, 5, 6, 7, 8 or all 9 substitutions compared to SEQ ID NO:16, selected from the group consisting of: (a) G18F, G18L, or G18M; (b) L50V; (c) V59L; (d) V72G; (e) M92W; (f) A96E or A96K; (g) L98V or L98Q; (h) E105W. Table 4 The polypeptide comprise multiple loop regions. In some embodiments, one or more loop residue relative to the reference polypeptide sequence may be substituted with any other amino acid. In another embodiment, amino acid residues in loop regions may be substituted with any other amino acid other than proline. In further embodiments, the loop regions may include additional amino acid residues relative to the reference polypeptide sequence, such as insertions of functional domains as described below. In some embodiments, any substitutions relative to the reference polypeptide sequence at amino acid residues in beta strand or alpha helical domains are conservative amino acid substitutions. As used herein, “conservative amino acid substitution” means a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Proteins comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, is retained. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non- conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into H is; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu. In another embodiment, any substitutions relative to the reference polypeptide sequence are conservative amino acid substitutions. In other embodiments, the polypeptides may comprise an insertion in a loop region, at the amino-terminus or the polypeptide, and/or at the C-terminus of the polypeptide. The polypeptide may comprise additional amino acid residues (“insertions”) as appropriate for an intended use. In non-limiting embodiments, such insertions may comprise leader sequences for secretion, tags for purification (including but not limited to His tags), detectable markers, and/or functional domain. By way of non-limiting examples, the functional domain may comprise a therapeutic polypeptide, a diagnostic polypeptide, a detectable polypeptide, a polypeptide component of a scaffold (such as a nanoparticle), a polypeptide to permit localization of the construct within a cell, tissue, or organism, etc. In another aspect, the disclosure provides kits comprising a combination of polypeptides, selected from the group consisting of: (a) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1; and (ii) a second polypeptide comprising or consisting of the amino acid sequence Gqrirvritg (SEQ ID NO:19); (b) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:2; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqricvritg (SEQ ID NO:20); (c) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1 or 4; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqrirvcitg (SEQ ID NO:21); (d) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1 or 4; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqrirvritg (SEQ ID NO:19); (e) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:5; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvtfivhe (SEQ ID NO:23); (f) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:6; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfqvre (SEQ ID NO:24); (g) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:7; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfnfre (SEQ ID NO:25); (h) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:8; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfnfre (SEQ ID NO:25); (i) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:9; and (ii) a second polypeptide comprising or consisting of the amino acid sequence sqthfevefkgmrirlrns (SEQ ID NO:27); (j) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:10; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gwlefeyekngrvirlvqg (SEQ ID NO:28); (k) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:11; and (ii) a second polypeptide comprising or consisting of the amino acid sequence sqtqfeyekngrrirlrqs (SEQ ID NO:29); and/or (l) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:12; and (ii) a second polypeptide comprising or consisting of the amino acid sequence ssvrveehmngvriqmeyg (SEQ ID NO:30). The polypeptides of SEQ ID NO:1-12 were designed to bind to the recited second polypeptides as recited above. These second polypeptides are capable for forming a self- associating ȕ-strand as described in the examples. The kits may be useful, for example, as heterodimers for various biotechnological applications. For instance for the delivery of genes too large for traditional adenoviral vectors; genes may be split in half and fused to each component of the heterodimer and reconstituted when expressed through the designed interface. Or for instance for covalently linking a therapeutic tagged with a designed peptide to the designed binders as a means of tethering a cargo to a transport molecule. In one embodiment, the first polypeptide comprises at least 4 alpha helices and at least 4 beta strands. In another embodiment, the first polypeptide comprises an alternating hydrophilic-hydrophobic side chain pattern. In another embodiment, binding pocket interface residues are conserved relative to the reference sequence. In a further embodiment, amino acid residues in loop regions of the first polypeptide may be substituted with any other amino acid. In one embodiment, any substitutions relative to the reference sequence at amino acid residues in beta strand or alpha helical domains of the first polypeptide are conservative amino acid substitutions. In another embodiment, any substitutions in the first polypeptide relative to the reference sequence are conservative amino acid substitutions. In one embodiment, the first polypeptide comprises an insertion in a loop region, at the amino- terminus or the polypeptide, and/or at the C-terminus of the first polypeptide. In another aspect the disclosure provides nucleic acids encoding the polypeptide of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA (such as an mRNA) or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure. In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence. "Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector. In another aspect, the disclosure provides host cells that comprise the nucleic acids, expression vectors (i.e.: episomal or chromosomally integrated), or polypeptides disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the nucleic acids or expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. In another aspect, the disclosure provides methods for treating an amyloid disease or amyloid-associated disease, comprising administering to a subject in need thereof an amount effective to treat the condition of the polypeptide of any embodiment or combination of embodiments herein. The disease may be any amyloid disease or amyloid-associated disease, including but not limited to Creutzfeldt-Jakob disease, spongiform encephalopathy, light chain amyloidosis, Huntington’s disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado-Joseph disease, Alzheimer’s disease, bovine spongiform encephalopathy, scrapie, type 2 diabetes, amyloidosis caused by transthyretin (ATTR), AA type amyloidosis, Parkinson’s disease, Lewy body disease, traumatic brain injury, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis-related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, multiple myeloma, and frontotemporal dementia. In one embodiment, the method comprises administering a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:13-18, 31-36 and 43- 45, wherein the polypeptide includes plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a self-associating ȕ- strand. As used herein, “treating an amyloid disease/amyloid-associated disease” means accomplishing one or more of the following: (a) reducing the severity of the disease; (b) limiting or preventing development of symptoms characteristic of the disease(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disease(s) being treated; (d) limiting or preventing recurrence of the disease(s) in patients that have previously had the disorder(s); (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disease(s); and (f) limiting development of the disease in a subject at risk of developing the disease, or not yet showing the clinical effects of the disease. As used herein, an “amount effective” refers to an amount of the polypeptide or multimer that is effective for treating and/or limiting amyloid disease or amyloid-associated disease. The polypeptides or multimers are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including orally, parentally, by inhalation spray, nasally, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra- arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. In one embodiment, administration is nasally. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). A suitable dosage range may, for instance, be 0.1 fg/kg-100 mg/kg body weight; alternatively, it may be 0.5 fg/kg to 50 mg/kg; 1 fg/kg to 25 mg/kg, or 5 fg/kg to 10 mg/kg body weight. The polypeptides or multimers can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician. In another aspect, the disclosure provides methods for diagnosing, prognosing, or monitoring an amyloid disease or amyloid-associated disease, comprising (a) contacting a tissue sample from a subject at risk of having an amyloid disease or amyloid-associated disease with the polypeptide of any embodiment herein, under conditions suitable for binding of the polypeptide with amyloid, if present in the tissue sample, to produce a binding complex (b) detecting binding complexes in the tissue samples; and (c) diagnosing or prognosing an amyloid disease or amyloid-associated disease based on the detecting. In one embodiment, the amyloid disease or amyloid-associated disease is selected from the group consisting of selected from the group consisting of Creutzfeldt-Jakob disease, spongiform encephalopathy, light chain amyloidosis, Huntington’s disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado-Joseph disease, Alzheimer’s disease, bovine spongiform encephalopathy, scrapie, type 2 diabetes, amyloidosis caused by transthyretin (ATTR), AA type amyloidosisParkinson’s disease, Lewy body disease, traumatic brain injury, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis-related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, multiple myeloma, and frontotemporal dementia. In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:13-18, 31-36, and 43-45, wherein the polypeptide includes plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a self-associating ȕ-strand. The methods of this aspect can be used to diagnose, prognose, or monitor a course of treatment for patients that may be suffering from an amyloid disease or amyloid-associated disease and to thus provide more informed determination of treatment options by an attending caregiver. Individuals at risk of an amyloid disease or amyloid-associated disease are those exhibiting one or more signs, symptoms, or risk factors for an amyloid disease or amyloid- associated disease, including but not limited to Creutzfeldt-Jakob disease, spongiform encephalopathy, light chain amyloidosis, Huntington’s disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado-Joseph disease, Alzheimer’s disease, bovine spongiform encephalopathy, scrapie, type 2 diabetes, amyloidosis caused by transthyretin (ATTR), AA type amyloidosis, Parkinson’s disease, Lewy body disease, traumatic brain injury, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis-related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, multiple myeloma, and diseases associated with amyloid-biofilm bacterial infections. The tissue sample may be any suitable tissue sample including, but not limited to blood, serum, cerebral spinal fluid, nasal secretions, urine or other biological material from a subject at risk of an amyloid disease or amyloid-associated disease. Any control may be used as determined appropriate by attending medical personnel. In one embodiment, the control may comprise a pre-determined level of binding complexes designated as “normal” (i.e.: not indicating the presence of amyloid disease or amyloid- associated disease). In another embodiment, the control may be a sample from a subject or subjects known not to have an amyloid disease or amyloid-associated disease. In other embodiments, the control may be a sample(s) from the same subject at one or more earlier time points; this embodiment is particularly useful for monitoring an amyloid disease or amyloid-associated disease, or monitoring efficacy of a treatment the subject is receiving for an amyloid disease or amyloid-associated disease. Detecting binding complexes in the tissue samples may be carried out by any suitable technique, including but not limited to labeling of the polypeptide with a detectable label (including but not limited to a fluorescent or luminescent protein) and detecting signal from the detectable label by a suitable technique, including but not limited to homogeneous and heterogeneous binding immunoassays, such as radioimmunoassays (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analyses. Examples Segments of proteins with ȕ-strand propensity can self associate to form amyloid fibrils associated with many diseases. These regions often adopt alternative structures in their folded states, or are intrinsically disordered in solution, making it difficult to generate binders or inhibitors with existing strategies. Here we describe a general approach to bind such segments in ȕ-strand and ȕ-hairpin conformations using de novo designed scaffolds that contain deep peptide binding clefts flanked by ȕ-strands that form hydrogen bonds to the peptide upon binding. The designs bind their cognate peptides in vitro with nanomolar affinities and in mammalian cells. The crystal structure of a designed protein-peptide complex is close to the design model. We use the approach to design binders to segments of the amyloid forming proteins Transthyretin, Tau, Serum amyloid A1 and Aȕ42 in order to block formation of amyloid oligomers and fibrils. Many proteins contain segments that only become ordered upon binding a target. (Tsai, Xu, and Nussinov 1998; Wright and Dyson 2009; Shammas et al.2016). A particularly interesting example of such disorder-to-order transitions are amyloidogenic sequences found in proteins such as Aȕ42, Tau and Serum amyloid A1. These regions can aggregate into amyloid fibrils via strand-strand interactions and are associated with amyloidosis and associated diseases both inside and outside the central nervous system (G.-F. Chen et al.2017; Gamblin et al.2003; Lu et al.2014; Iakovleva et al.2021; Knowles, Vendruscolo, and Dobson 2014; Chiti and Dobson 2006). Designed binders to amyloid forming segments of these proteins could have utility both as diagnostics and therapeutics. However, it is difficult to raise antibodies against the monomeric form of amyloid due to their strong tendency for self association; this also complicates the systematic generation of binders using library selection methods. The multiplicity of conformations complicates design of binders to disordered protein segments, and the computational design of binders to amyloid forming segments of proteins remains an outstanding challenge. We reasoned that this challenge could be overcome by taking advantage of the ȕ- strand forming propensity of amyloidogenic peptides. To design binders to peptides in extended ȕ-strand conformations, we sought to create scaffolds that could provide ȕ-strand pairing interactions to all the backbone amide and carbonyl atoms of the peptide such that the peptide strand complements a ȕ-sheet on the scaffold (Fig 1A). Starting from Fold-It^TM designed proteins with mixed Į/ȕ topology (Koepnick et al.2019), we designed additional strands and helices to create scaffolds with a single central ȕ-strand missing from an extended ȕ-sheet. The sheet is buttressed by Į-helices which pack on one another to support the structure in the absence of the bound peptide (see Fig 1b and Methods). Rosetta ^TM combinatorial sequence design calculations were then used to optimize the sequences of both the scaffold and the peptide for high affinity binding (we reasoned that such “two-sided” designs would be an easier starting point than “one sided” designs against amyloid forming peptides where only the sequence of the binders are allowed to be optimized). Designs with favorable interaction energy, few unsatisfied buried polar atoms and high shape complementarity, and for which Rosetta ^TM structure predictions were close to the designed scaffold and complex structures were selected for experimental characterization. The selected designs without their cognate peptides were encoded in synthetic genes with an N-terminal polyhistidine affinity tag, expressed in Escherichia coli, and purified using immobilized nickel affinity chromatography (IMAC) followed by size exclusion chromatography (SEC). Despite the absence of the peptide, a large number of designs expressed well and were monodisperse in SEC. Bicistronic vectors were generated for each of the monodisperse designs; the first cistron encodes sfGFP fused at its C-terminus to the designed peptide, and the second cistron the polyhistidine tagged designed binder. After expression of the bicistronic constructs, binding of the GFP-peptide fusion to the his-tagged binder was assessed by SDS-PAGE following purification by IMAC and SEC. The binding of six designs (figure 2 and table 5) that were well expressed, soluble, and monodisperse by SEC, to their designed peptide targets was further characterized using biolayer interferometry (BLI) by immobilizing chemically synthesized biotinylated peptides on streptavidin sensors and dipping these into a solution with the designed binding partner. The interaction kinetics ranged from 10⁴ M^-1 s^-1 to 10² M^-1 s^-1 for association and between 0.17 s^-1 - 10^-4 s^-1 for the dissociation (table 6). The equilibrium dissociation constant KD ranged from 44 ^M to 150 nM with no clear distinction between single strand and hairpin binders (table 6). For design C104 we confirmed binding in an orthogonal SEC binding assay (figure 8a). Single amino acid substitution of the buried residue Val6 in the peptide of C104 to Arg completely disrupted binding in BLI suggesting that the designed binding mode is recapitulated (figure 8b and 8c). Table 5. Overview designs The designed peptides are amphipathic with an alternating hydrophilic-hydrophobic side chain pattern (figure 8d). Beyond the backbone ȕ-strand hydrogen bonding, the peptide- binder interaction consists of somewhat separable solvent exposed and solvent shielded interfaces. The solvent inaccessible part of the interface consists primarily of the hydrophobic residues that closely pack against the hydrophobic core of the binder and drive the association between peptide and binder (figure 3a, top). In design CH17 these interactions are accompanied by designed buried hydrogen bond networks (figure 3b) (Boyken et al.2016). The solvent exposed portion of the interface (figure 3a, bottom) is composed primarily of salt bridges and hydrogen bonds that likely make less of a contribution to the overall interface energy because of competition with water. Because the hydrophobic-hydrophilic patterning is shared among the designed peptides, not all designs are able to fully discriminate between cognate and non-cognate peptides enabling them to sequester a broad range of peptides (data not shown) that have similar physicochemical properties. Design CH17 that contains buried hydrogen bond network is however more selective to its cognate peptide, because binding of a non-complementary peptide would bury polar residues that are not satisfied with a hydrogen bond donor/acceptor disfavoring binding. We explored the possibility of increasing peptide binding affinity by introducing hydrophobic interaction pairs across solvent exposed parts of the interface using combinatorial side chain design in Rosetta^TM. Introduction of an exposed hydrophobic interaction pair in design C34.1 improved the K_D 6-fold to 2 ^M from 12 ^M in parent design C34 (figure 3c and table 6). In CH15.1 we introduced 3 hydrophobic interaction pairs that when combined led to a 400-fold improvement of the K_D from 40 ^M to 100 nM compared to the parent CH15 design (figure 9a and 9b and table 6). The modified designs remained monomeric, indicating that these surface substitutions are generally well tolerated (figure 9c and 9d). Disulfide functionalization could enable redox control of binding activity for a variety of biotechnological applications. We searched for positions that could host a disulfide bridge across the interface of C104 using the Disulfidize mover in Rosetta ^TM (Fleishman et al.2011; Bhardwaj et al.2016) and found several positions where low energy disulfides could be modeled (figure 4a and figure 10a). For designs C104.2 and C104.3 we confirmed through non-reducing SDS-PAGE analysis that disulfides indeed formed (figure 4b and figure 10b). For C104.3 this result was further validated in a SEC subunit exchange experiment where we first reconstituted the non-covalent complex between C104 and its peptide fused to the c- terminus of ubiquitin, as well as the disulfide linked complex between C104.3 and its cysteine containing peptide fused to c-terminus ubiquitin. When the preformed non-covalent complex was mixed with GFP-104 and ran over SEC, GFP-104 co-eluted with C104 as observed through the absorbance at 395 nm indicating GFP-104 could exchange with ubiquitin-peptide fusion to bind C104 (figure 4c). In contrast, the peptide in the covalent C104.3 complex could not be outcompeted when it was mixed with GFP-P104 due to the disulfide bridge (figure 4d and figure 10b). Small peptides are useful as affinity tags to bind and localize tagged protein partners into larger molecular assemblies. To demonstrate the utility of our designs towards such applications and also for use in novel customizable protein materials, we rigidly fused binder C37 to the LHD284B9 component of the LHD hetero-oligomer system that consist of de novo designed protein building blocks that can be assembled into a large variety of multiprotein complexes. Fusion creates single chain proteins with two different interfaces; one peptide binding interface and one LHD heterodimer interface. Mixing for instance GFP tagged peptide of C37 with C37LHD284B9 creates a heterodimer. This assembly can further be expanded by the addition of for example LHD284A82 creating a heterotrimer. We confirmed the assembly of this complex via SEC (figure 11). We obtained a 2.3 Å resolution crystal structure of a variant of C104, C104.1, where all the surface residues outside the interface were redesigned using ProteinMPNN. The crystal structure recapitulates the designed model with both individual domains clamping the peptide in a ȕ-strand conformation (figure 5 and table 7). The individual domains superimpose well with the design model. The majority of the peptide is resolved in the electron density and binds in a ȕ-strand conformation with the apolar residues buried in the designed cleft (figure 5). A deviation from the designed model at helix3 shifts Tyr91 towards the peptide-binding pocket in the crystal structure partially occluding it (figure 5). As a result peptide residue Ile8 is displaced and the last few residues of the peptide are disordered in the crystal and not modeled (see methods). Table 7. Crystallographic data collection and refinement. While we were not able to obtain a crystal structure of a hairpin binding design, strand deletion experiments support that these peptides bind to the scaffold in a hairpin conformation rather than through single strand insertion: the binding of each individual strand of the CH15.1 hairpin to the CH15.1 binder is weaker than binding of the whole hairpin by BLI (data not shown). Encouraged by the biochemical and structural validation of our design approach on the two sided binder design challenge, we next investigated whether the approach could generate binders to naturally occurring peptide or protein segments which form amyloids in a range of disease states. This is a more challenging “one sided” design problem because the target sequence is fixed. Amyloid fibril deposits can form in the central nervous system as is the case for Aȕ42, Microtubule associated protein Tau, and alpha-synuclein but also extra- cerebrally like in transthyretin and serum amyloid A1 mediated amyloidosis (Lu et al.2014; Bloom 2014; Muchtar et al.2021). The fibrils form through strand-strand mediated oligomerization/fibrillization and are harmful to cells and tissues (Knowles, Vendruscolo, and Dobson 2014; Chiti and Dobson 2006). We aimed to design binders to fibril forming regions to block or modulate fibril assembly (figure 6). To design such binders, we started from the design constraint that the peptide side chains facing the core of the binding scaffold must be primarily hydrophobic; since the peptide is bound in a ȕ-strand conformation, every other residue is in the core and hence must be hydrophobic. We scanned the primary sequences of the Abeta peptide, Microtubule associated protein Tau, transthyretin and serum amyloid A1 for regions that matched this pattern (fig 12). Matched regions were docked in a ȕ-conformation into the binding cleft of the scaffolds, and the scaffold interface residues were redesigned to maximize contacts to the amyloid derived ȕ-strand, including surface-exposed hydrophobic interactions as described above. Designs with docked peptides predicted to participate in fibril or oligomer formation based on experimentally determined amyloid structures were selected for experimental characterization. The amyloid strand binders were first tested using the bicistronic expression screen described above; amyloid peptide fragments were fused to the C-terminus of GFP and co- expressed with polyhistidine tagged binder. After IMAC purification, we found using SDS PAGE that peptides derived from Aȕ42, Transthyretin, Tau and Serum amyloid A1 interacted with the binders. In SEC, binder and peptide fusion protein co-eluted indicating the complexes remain stably associated even when diluted on the column. The designed scaffolds were also stable and mostly monodisperse by SEC when purified in absence of their target peptides. We synthesized biotinylated versions of the single strand Aȕ42, Transthyretin, Tau and Serum amyloid A1 fragments targeted by the designs and immobilized them on streptavidin biosensors to test in BLI. All purified designs bound their target peptides (figure 7); we also observed some cross-reactivity consistent with similarities in the amyloid forming sequences. For example, the Aȕ42 binders DAm14 and DAm15 bind their target and also interact with peptides derived from Transthyretin and Tau. Circular dichroism spectroscopy and SEC experiments indicated DAm14 and DAm15 were folded and thermostable, indicating that the promiscuous binding was not due to protein unfolding (data not shown). Other designs such as the transthyretin binder DTTR23, Tau binder 2DT2 and serum amyloid A binder DSAA1_1 were more selective towards their targets (data not shown). To increase the affinity, site saturation mutagenesis (SSM) was performed on designs HLTAU_014, 2HLTAU_011, and DAm_015. Analysis of the SSMs showed high conservation of binding motifs. Residue substitutions that showed enrichment across the SSM experiments were selected for testing. For HLTAU_014_v2 and HLTAU_014_v8, there was a 10-fold decrease in KD indicating tighter binding affinity (data not shown). For remaining substitutions indicated in the tables, binding affinity was similar to the original. The positions in the scaffold outside the interface are required for proper folding of the design and hence binding pocket. These positions can be divided into structurally essential and structurally non-essential positions (data not shown). In the essential positions there is limited tolerance for substitutions. These positions are generally in the interior of the protein or at certain positions in loops. The other non-essential positions are generally solvent exposed and have more tolerance with respect to substitutions. After characterizing the binding interaction between the binders and their targets the effect of the designs on amyloid fibril formation was studied. Aȕ42 fibril formation was tested in the presence of DAm_012, DAm_014 and DAm_015 in a Thioflavin T (ThT) assay. Robust fibril formation was observed in the control reactions but in presence of the designs fibril formation was significantly retarded in a concentration dependent manner, with DAm_012 and DAm_014 being more potent than DAm_015 (data not shown). DAm14 and DAm15, at stoichiometric ratios, completely inhibited fibril growth for at least 30h. DAm_012 prevented detectable amyloid formation for 10h even under a 1:2 sub- stoichiometric ratio of inhibitor to peptide, comparable to clinical stage therapeutic antibodies raised against this same target, including one approved drug, aducanumab (data not shown). Like DAm14 and DAm15, DAm12 was thermostable and remained folded up to 94°C in CD spectroscopy. In a control experiment, C104 and a previously de novo designed binder with a mixed Į/ȕ topology showed significantly lower inhibitory potential, indicating that the presence of a hydrophobic cleft surrounded by ȕ-sheet structure is insufficient for inhibition (data not shown). For the Aȕ42 target, we in addition to binders that bind a small fragment of Aȕ42 as a single strand, also designed proteins that bind a longer Aȕ42 fragment as a beta hairpin (Table 5). Three designs designated DABxtalM3, DABxtalM5, and DABxtalM9 (SEQ ID NO:43-45) strongly bound their targets in bicistronic screening assays (data not shown). Discussion We present a general approach for designing binders targeted to disordered stretches of proteins and peptides that can adopt ȕ-strand or ȕ-hairpin conformations. The designed binders are folded and bind the target peptides with nanomolar affinities in vitro and in cells and can be incorporated into larger assemblies through fusion of peptide or binder to other components. Binding hydrophobic regions of proteins is challenging because the properties that make proteins stick to hydrophobic surfaces can also lead to poor solubility and highly indiscriminate binding; the overall geometry of the designed binding pocket and possible dynamic sheet opening/closure appear to limit such adverse effects. While the apo state is likely dynamic, the x-ray crystal structure of a designed binder-peptide complex is highly ordered and very close to the design model. The highly specific shape complementary binding pockets in our designs nearly completely engulf the bound peptide. This enables capture of protein segments that are prone to amyloid formation such as those found in Amyloid precursor protein, Microtubule associated protein Tau, transthyretin and serum amyloid A1. The designs potently inhibit the formation of the Aȕ42 fibrils that are a hallmark of Alzheimer's disease (AD), at a similar potency as clinically evaluated antibodies, including an approved drug (aducanamab). This result is particularly significant since it is challenging to elicit antibodies to monomeric forms of peptides which spontaneously self associate, and structurally our ȕ-sheet clamping approach can likely generate more extensive interactions with extended ȕ-strand peptides than antibody loops. The designs are also useful in blocking smaller amyloidogenic oligomers, as the oligomers use similar stretches of sequences to self-assemble and are considered highly toxic precursors to fibrils. The designs are useful as diagnostics and therapeutics for treating AD and other amyloid diseases. Materials and methods Protein design Backbone generation We explored two approaches to generate scaffolds with ȕ-sheets with open slots for peptide ȕ-strand insertion (figure 1b and 8) using blueprint based backbone building in (Py)Rosetta (Koga et al.2012; Lin et al.2015; Huang et al.2011; Chaudhury, Lyskov, and Gray 2010; Leman et al.2020) . In a first two-domain binder approach (figure 8a), we started from a scaffold, 2003285_0000, designed by Fold-It players (Koepnick et al.2019) (domain 1) and generated a ȕ-sheet that extends from the C-terminal strand of the scaffold using blueprint based backbone generation (Huang et al.2011; Koga et al.2012). In the next step this sheet was further expanded into a second mixed alpha/beta domain with three strands and one helix or four strands and two helices. The central strand of the ȕ-sheet that encompasses both domains was split off from generating an individual peptide in ȕ-strand conformation that can bind the designed deep cleft between domain 1 and domain 2. A connecting loop linking the helices that make up the interdomain interface was next generated using loop closure (Brunette et al.2015) to yield a single polypeptide two-domain binder that clamps the peptide on either side through ȕ-strand backbone hbonds (figure 8a). The same approach was followed to generate ȕ-hairpin binding scaffolds. In the second approach, a different foldit scaffold, 2003333_0006 (Koepnick et al. 2019), was modified to function as a peptide binder (figure 8b). The connection between ȕ- strand 3 and 4 was removed to create the individual peptide component. To stabilize the modified binder and ensure its solubility in absence of the peptide, we designed buttressing secondary structure elements that support the binding interface and scaffold. ȕ-strand 3 was paired with another antiparallel strand whereas helices 1 and 2 were backed up by either one or two supporting helices. After backbone generation, Rosetta^TM combinatorial sequence design calculations were used to optimize the sequences of both the scaffold and the peptide for high affinity binding. Designs with favorable interaction energy, few unsatisfied buried polar atoms and high shape complementarity, and for which Rosetta^TM folding simulations yielded models close to the designed model were selected for experimental characterization. Sequence design The amino acid sequence of the newly built polyvaline backbones were optimized using Rosetta^TM flexible backbone enabled combinatorial side chain design followed by a second design round for the peptide-binder interface. Ref2015, beta_nov16 or beta_genpot scorefunctions were used during design (Alford et al.2017). For a subset of designs, buried polar hydrogen bond networks were designed using the HBNet^TM mover (Boyken et al. 2016). The affinity between peptide and binder was computationally improved by introducing hydrophobic interaction pairs to the solvent exposed side of the interface. All solvent exposed interactions pairs for which the CĮ atoms were within 6 Å from each other were selected and allowed to be redesigned with the PackRotamersMover^TM to only Phe, Ala, Met, Ile, Leu, Tyr, Val and Trp using a fixed backbone. For the computational affinity optimization of the natural target peptides, all surface exposed residues on only the binder within 6 Å of the target hydrophobic side chain were allowed to be redesigned. Residues around the redesigned interactions pairs were repacked. Single redesigned pairs and combinations of pairs were selected for experimental characterization. In order to facilitate crystallization, the surface residues outside the interface were redesigned using ProteinMPNN for design C104. The structure of sequences obtained from ProteinMPNN were predicted using AlphaFold2 (Jumper et al.2021) and designs with rmsd <= 1.5 and plDDT >= 85 to the original designed model were selected for experimental characterization. Design of rigid helical fusions Rigid fusions of peptide binders and components of the LHD hetero-oligomer system was performed as described previously (Hsia et al.2021; Sahtoe et al.2022). Matching natural peptide sequences to scaffolds The protein sequences of Amyloid precursor protein, Microtubule associated protein Tau, Transthyretin and Serum amyloid A1 were searched for burial patterns that are also present in the peptides of designs C34, C37, C104 and CH15. For C104 both the designed model and the crystal structure of C104, minimized with FastRelax^TM (Tyka, Jung, and Baker 2012), was used. The burial patterns representing relative positions of solvent inaccessible residues versus solvent accessible residues in the designed peptides were identified by visual inspection. For each peptide, all amyloidogenic protein sequence-frames of length n, where n is the number of residues in the designed peptide, were scanned for matching regions. Only residues Phe, Ala, Met, Ile, Leu, Val or Gly were allowed at the solvent inaccessible positions. At the remaining positions, all residues were allowed except for Pro which was only allowed at either terminus. When a match was identified, the sequence of the template designed peptide was mutated to the sequence of the matched sequence of the amyloidogenic protein. The resulting peptide-binder complex was minimized and the residues in the interface of the designed binder were redesigned to optimally match the amyloidogenic sequence by also including hydrophobic interaction pairs across the solvent accessible area of the interface (see above). Protein expression and purification Synthetic genes encoding designed proteins were purchased from Genscript or Integrated DNA technologies (IDT) in the pET29b expression vector or as eBlocks^TM (IDT) and cloned into customized expression vectors (Wicky et al.2022) using golden gate cloning. A His6x tag was included either at the N-terminus or at the C-terminus as part of the expression vector. In some cases a TEV protease recognition site was introduced at the N- terminus after the histidine tag. Peptide genes were purchased as fusion proteins to either the C-terminus of sfGFP or the N-terminus of a ubiquitin-AviTag-His6x construct separated by a Pro-Ala-Ser linker. Bicistronic genes were ordered as described (Sahtoe et al.2022). Detailed construct information is provided in the supplementary information. Proteins were expressed using autoinducing media consisting of TBII media (Mpbio) supplemented with 50x5052, 20 mM MgSO4 and trace metal mix in BL21 LEMO E.coli cells. Proteins were expressed under antibiotic selection at 37 degrees Celsius overnight or at 18-25 degrees Celsius overnight after initial growth for 6-8h at 37 degrees Celsius. Cells were harvested by centrifugation at 4000x g and resuspended in lysis buffer (100 mM Tris pH 8.0, 200 mM NaCl, 50 mM Imidazole pH 8.0) containing protease inhibitors (Thermo Scientific) and Bovine pancreas DNaseI (Sigma-Aldrich) before lysis by sonication. One millimolar of the reducing agent TCEP was included in the lysis buffer for designs with free cysteines. Proteins were purified by Immobilized Metal Affinity Chromatography. Cleared lysates were incubated with 2-4ml nickel NTA beads (Qiagen) for 20-40 minutes before washing beads with 5-10 column volumes of lysis buffer, 5-10 column volumes of high salt buffer (10 mM Tris pH 8.0, 1 M NaCl) and 5-10 column volumes of lysis buffer. Proteins were eluted with 10 ml of elution buffer (20 mM Tris pH 8.0, 100 mM NaCl, 500 mM Imidazole pH 8.0). His6x tags were cleaved by dialyzing IMAC elutions against 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM TCEP overnight in the presence of His6x tagged TEV protease followed by a second IMAC column to remove His6x-TEV and uncleaved protein. Single cysteine variants of DAm12, DAm14 and DAm15 where purified as described above and labeled with Alexa^TM 488-C5-maleimide (Thermo) at a concentration of between 50-100 ^M of protein and a 2-5 fold molar excess of label in SEC buffer supplemented with 1 mM TCEP protected from light. After 3h at room temperature or overnight at 4 degrees Celsius the labeling reaction was quenched by the addition of 1M DTT. All protein preparations were as a final step polished using size exclusion chromatography (SEC) on either Superdex^TM 200 Increase 10/300GL or Superdex^TM 75 Increase 10/300GL columns (Cytiva) using 20 mM Tris pH 8.0, 100 mM NaCl. The reducing agent TCEP was included (1 mM final concentration) for designs with free cysteines. For designs where a substantial void volume peak was present in addition to the monomer peak, the monomer peak was pooled and reinjected. Only designs where upon reinjection the void peak was mostly absent were further pursued. SDS-PAGE and LC/MS were used to verify peak fractions. Proteins were concentrated to concentrations between 0.5-10 mg/ml and stored at room temperature or flash frozen in liquid nitrogen for storage at -80. Thawing of flash frozen aliquots was done at room temperature or 37 degrees Celsius. All purification steps from IMAC were performed at ambient room temperature. The C104.1 complex was prepared by incubating binder with a 3-5 fold molar excess of the peptide for 3h at room temperature followed by SEC. Peptide synthesis All Fmoc-protected amino acids were purchased from P3 Bio. Oxyma was purchased from CEM; DIC from Oakwood Chemicals. DMF was purchased from Fisher Scientific and treated with an Aldraamine^TM trapping pack (Sigma-Aldrich) prior to use. Piperidine was purchased from Sigma-Aldrich. Cl-TCP(Cl) resins were purchased from CEM. The peptides were synthesized on a 0.1mmol scale using microwave-assisted solid-phase peptide synthesis via a CEM LibertyBlue^TM system, then subsequently cleaved with a cleavage cocktail consisting of TFA, TIPS, water, and DODT (92.5:2.5:2.5:2.5 in order). The cleavage solution was concentrated in vacuo, precipitated into cold ether, and spun down by way of centrifugation. This pellet was washed and spun down again with ether (2x), then dried under nitrogen, resuspended in water and ACN, and purified by RP-HPLC on an Agilent 1260 Infinity Semi-prep system with a gradient from 20% to 70% over a period of 15min (A: H2O with 0.1% TFA, B: ACN with 0.1% TFA). The purified peptide fractions were combined into one, lyophilized, and massed in a tared scintillation vial for the final product. Peptides derived from Transthyretin, Tau, and Serum amyloid A1 were purchased from WuXi. Depending on the isoelectric point, lyophilized peptides were solubilized in buffers containing either 100 mM Tris pH 8.0 or 100 mM MES pH 6.5 and stored at -20 degrees Celsius. Mammalian cell culture and transfection HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 1 mM L-glutamine (Gibco), 4.5 g/liter D-glucose (Gibco), 10% fetal bovine serum (FBS), and (1×) nonessential amino acids (Gibco). Cells were kept in culture at 37°C and 5% CO2 and split twice per week by trypsinization using 0.05% trypsin EDTA (Gibco) followed by passage at 1:5 or 1:10 into a new tissue culture (TC)–treated T75 flask (Thermo Scientific ref 156499). Before transfection, cells were plated at 20,000 cells per well in Cellview^TM cell culture slides (Greiner Bio-One ref 543079) for 24 hours after which transfection took place using 187.5 ng total DNA per well and 1 ^g/^l PEI- MAX^TM (Polyscience) mixed with Opti-MEM^TM medium (Gibco). Transfected cells were incubated at 37°C and 5% CO2 for 24 to 36 hours before being imaged. Fluorescent microscopy Three dimensional images were acquired with a commercial OMX-SR system (GE Healthcare) using a 488 nm Toptica diode laser for excitation. Emission was collected on a PCO.edge sCMOS cameras using an Olympus 60× 1.42NA PlanApochromat^TM oil immersion lens.1024×1024 images (pixel size 6.5 ^m) were captured without binning. AcquireSR^TM Acquisition control software was used for data collection. Z-stacks were collected with a step size of 500 nm and 15 slices per image. The images were deconvolved with an enhanced ratio using SoftWoRx^TM 7.0.0 (GE Healthcare). Finally, cell images were sum projected using Fiji v2.1.0. Scale bars equal 10 microns. Biolayer interferometry Biolayer interferometry experiments were performed on an OctetRED^TM96 BLI system (ForteBio, Menlo Park, CA) at room temperature in Octet^TM buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) supplemented with 1mg/ml bovine serum albumin (SigmaAldrich). Prior to measurements, streptavidin-coated biosensors were first equilibrated for at least 10 min in Octet^TM buffer. Chemically synthesized peptides with a C-terminal biotin or enzymatically biotinylated peptide-fusion proteins (see supplementary spreadsheet for details) were immobilized onto the biosensors by dipping them into a solution with 100 to 500 nM protein until the response reached between 10% and 50% of the maximum value followed by dipping sensors into fresh octet buffer to establish a baseline for 60 s. Titration experiments were performed at 25 degrees^Celsius while rotating at 1000 rpm. Association of designs was allowed by dipping biosensors in solutions containing designed protein diluted in Octet^TM buffer until equilibrium was approached followed by dissociation by dipping the biosensors into fresh buffer solution to monitor the dissociation kinetics. In the peptide binding cross specificity assays each biotinylated peptide was loaded onto streptavidin biosensors in equal amounts followed by 2 min of baseline equilibration. Then association and dissociation with all the different binders was allowed for 400 s for each step. For the designed peptide-binder pairs, binder concentrations were around the Kd of the interaction between the loaded peptide and its designed binding partner whereas the concentrations for the amyloid binders were 10, 2.5 and 0.625 ^M. Global kinetic or steady-state fits were performed on buffer subtracted data using the manufacturer’s software (Data Analysis 9.1) assuming a 1:1 binding model. Enzymatic biotinylation of proteins Proteins with Avi-tags (GLNDIFEAQKIEWHE (SEQ ID NO:41); see supplementary materials) were purified as described above and biotinylated in vitro using the BirA500 (Avidity, LLC) biotinylation kit.840 ul of protein from an IMAC elution was biotinylated in a 1200 ^l (final volume) reaction according to the manufacturer’s instructions. Biotinylation reactions were allowed to proceed at either 4°C overnight or for 2-3 hours at room temperature on a rotating platform. Biotinylated proteins were purified using SEC on a Superdex^TM 20010/300 Increase GL (GE Healthcare) or S7510/300 Increase GL (GE Healthcare) using SEC buffer (20 mM Tris pH 8.0, 100 mM NaCl). Circular Dichroism Spectroscopy CD spectra were recorded in a 1 mm path length cuvette at a protein concentration between 0.3-0.5 mg/mL on a J-1500 instrument (Jasco). For temperature melts, data were recorded at 222 nm between 4 and 94 °C every 2 C°, and wavelength scans between 190 and 260 nm at 10 C° intervals starting from 4 C°. Experiments were performed in 20 mM Tris pH8.0, 20 mM NaCl. The high tension (HT) voltage was monitored according to the manufacturer’s recommendation to ensure optimal signal-to-noise ratio for the wavelengths of interest. SEC binding assays SEC binding assays between purified designs and GFP-peptide fusions were performed on a Superdex^TM 75 increase 10/300 GL (Cytiva) in 20 mM Tris pH 8.0, 100 mM NaCl using 500 ul injections containing 15 or 20 ^M final concentration of each component. Binding reactions were allowed to equilibrate for at least 45 minutes before injection. For the subunit exchange experiment, the disulfide stabilized complex between C104.2 and ubiquitin- pep104.2 as well as the control base non-covalent complex were allowed to form overnight at a 20 ^M equimolar concentration under oxidizing conditions after which competing GFP- pep104 was added to the pre-formed complexes to a final concentration of 20 ^M. After at least 45 minutes the reaction was injected on SEC. Elution profiles were collected by monitoring absorbance at 230 nm and 395 nm (absorbance of GFP). All experiments were performed at room temperature. Disulfide formation assay Individual protein components were purified as described above in the presence of 1 mM TCEP except for in the last SEC step where no reducing agent was present. Reactions were incubated at room temperature using 50 ^M of each component in 20 mM Tris pH 8.0, 100 mM NaCl. Reactions were stopped by adding an equal volume of 2x non-reducing SDS protein loading buffer at the indicated time points. Crystal structure determination The C104.1 complex was (19 mg/ml) crystallized using the vapor diffusion method at room temperature in 0.1 M Tris pH 7.8, poly-Ȗ-glutamic acid low molecular weight polymer, 15% PEG 4000 (Molecular dimensions) before the crystals were harvested in 25% glycerol as a cryoprotectant. Data was collected at the Advanced Photon Source at Argonne National Laboratory. Diffraction images were integrated using XDS (Kabsch 2010) or HKL3000 (Otwinowski and Minor 1997) and merged/scaled using Aimless (Winn et al.2011). Starting phases were obtained by molecular replacement using Phaser (McCoy et al.2007) using the computational design models of the individual N and C terminal domains of C104.1 as search models. Structures were refined using either phenix.refine (Adams et al.2010) or Refmac (Murshudov, Vagin, and Dodson 1997) and PDB-REDO (Joosten et al.2014). Model building was performed using COOT (Emsley and Cowtan 2004). Lack of density at the C- terminus of the peptide prompted us to examine the possibility of a ȕ-strand register shift for the peptide binding. OMIT maps were used to decrease the model bias. In addition, the peptide was modeled in several off-target ȕ-strand registers. Overall refinement statistics and B-factors, were better for the model where the peptide was modeled in the designed on-target ȕ-strand register. The final model was evaluated using MolProbity (Williams et al.2018). Data collection and refinement statistics are recorded in Table 7. 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Claims

We claim 1. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:1-18, 31-36, and 43-45, wherein the polypeptide includes a plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a ȕ-strand.

2. The polypeptide of claim 1, wherein the polypeptide comprises at least 4 alpha helices and at least 4 beta strands.

3. The polypeptide of claim 1 or 2, wherein any substitution of binding pocket interface residues is a conservative amino acid substitution relative to the reference sequence.

4. The polypeptide of any one of claims 1-3, wherein binding pocket interface residues are conserved relative to the reference sequence.

5. The polypeptide of any one of claims 1-4, wherein amino acid residues in loop regions may be substituted with any other amino acid; optionally wherein amino acid residues in loop regions may be substituted with any other amino acid other than proline.

6. The polypeptide of any one of claims 1-5, wherein any substitutions relative to the reference sequence at amino acid residues in beta strand or alpha helical domains are conservative amino acid substitutions.

7. The polypeptide of any one of claims 1-6, wherein any substitutions relative to the reference sequence are conservative amino acid substitutions.

8. The polypeptide of any one of claims 1-7, comprising an insertion in a loop region, at the amino-terminus or the polypeptide, and/or at the C-terminus of the polypeptide.

9. The polypeptide of claim 1, wherein the polypeptides comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:31, 35, and 36, wherein the polypeptide comprises 1, 2, 3, 4, or all 5 substitutions compared to SEQ ID NO:31, 35, or 36 selected from the group consisting of: (a) V4I or V4Y; (b) L6V; (c) R11T or T11R; (d) H83D, H83N, or N83H; and (e) H122L.

10. The polypeptide of claim 1, wherein the polypeptides comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:33, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6 or all 7 substitutions compared to SEQ ID NO:33, selected from the group consisting of: (a) V6Y or V6W; (b) I11M; (c) A41Y; (d) Q44E; (e) P80R or P80A; (f) R106N or R106Q; and (g) M107W.

11. The polypeptide of claim 1, wherein the polypeptides comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:16, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8 or all 9 substitutions compared to SEQ ID NO:16, selected from the group consisting of: (a) G18F, G18L, or G18M; (b) L50V; (c) V59L; (d) V72G; (e) M92W; (f) A96E or A96K; (g) L98V or L98Q; (h) E105W.

12. A kit comprising a combination of polypeptides, selected from the group consisting of: (a) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1; and (ii) a second polypeptide comprising or consisting of the amino acid sequence Gqrirvritg (SEQ ID NO:19); (b) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:2; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqricvritg (SEQ ID NO:20); (c) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1 or 4; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqrirvcitg (SEQ ID NO:21); (d) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:1 or 4; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gqrirvritg (SEQ ID NO:19); (e) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:5; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvtfivhe (SEQ ID NO:23); (f) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:6; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfqvre (SEQ ID NO:24); (g) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:7; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfnfre (SEQ ID NO:25); (h) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:8; and (ii) a second polypeptide comprising or consisting of the amino acid sequence dvrfnfre (SEQ ID NO:25); (i) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:9; and (ii) a second polypeptide comprising or consisting of the amino acid sequence sqthfevefkgmrirlrns (SEQ ID NO:27); (j) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:10; and (ii) a second polypeptide comprising or consisting of the amino acid sequence gwlefeyekngrvirlvqg (SEQ ID NO:28); (k) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:11; and (ii) a second polypeptide comprising or consisting of the amino acid sequence sqtqfeyekngrrirlrqs (SEQ ID NO:29); and/or (l) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence of SEQ ID NO:12; and (ii) a second polypeptide comprising or consisting of the amino acid sequence ssvrveehmngvriqmeyg (SEQ ID NO:30).

13. The kit of claim 12, wherein the first polypeptide comprises at least 4 alpha helices and at least 4 beta strands.

14. The kit of claim 12 or 13, wherein the first polypeptide comprises an alternating hydrophilic-hydrophobic side chain pattern.

15. The kit of any one of claims 12-14, wherein binding pocket interface residues are conserved.

16. The kit of any one of claims 12-15, wherein amino acid residues in loop regions of the first polypeptide may be substituted with any other amino acid.

17. The kit of any one of claims 12-16, wherein any substitutions relative to the reference sequence at amino acid residues in beta strand or alpha helical domains of the first polypeptide are conservative amino acid substitutions.

18. The kit of any one of claims 12-17, wherein any substitutions in the first polypeptide relative to the reference sequence are conservative amino acid substitutions.

19. The kit of any one of claims 12-18, comprising an insertion in a loop region, at the amino-terminus or the polypeptide, and/or at the C-terminus of the first polypeptide.

20. A nucleic acid encoding the polypeptide as recited in any preceding claim.

21. An expression vector comprising the nucleic acid of claim 20 operatively linked to a suitable control element.

22. A recombinant host cell comprising the polypeptide, kit, nucleic acid, and/or expression vector of any preceding claim.

23. A method for treating an amyloid disease or amyloid-associated disease, comprising administering to a subject in need thereof an amount effective to treat the condition of the polypeptide of any one of claims 1-11.

24. The method of claim 23, wherein the amyloid disease or amyloid-associated disease is selected from the group consisting of selected from the group consisting of Creutzfeldt-Jakob disease, spongiform encephalopathy, light chain amyloidosis, Huntington’s disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado-Joseph disease, Alzheimer’s disease, bovine spongiform encephalopathy, scrapie, type 2 diabetes, amyloidosis caused by transthyretin (ATTR), AA type amyloidosis, Parkinson’s disease, Lewy body disease, traumatic brain injury, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis- related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, multiple myeloma, and frontotemporal dementia.

25. The method of claim 23 or 24, wherein the method comprises administering a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:13-18, 31-36, and 43-45, wherein the polypeptide includes plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a self-associating ȕ-strand.

26. A method for diagnosing, prognosing, or monitoring an amyloid disease or amyloid- associated disease, comprising (a) contacting a tissue sample from a subject at risk of having an amyloid disease or amyloid-associated disease with the polypeptide of any one of claims 1-8, under conditions suitable for binding of the polypeptide with amyloid, if present in the tissue sample, to produce a binding complex (b) detecting binding complexes in the tissue samples; and (c) diagnosing or prognosing an amyloid disease or amyloid-associated disease based on the detecting.

27. The method of claim 26, wherein the amyloid disease or amyloid-associated disease is selected from the group consisting of selected from the group consisting of Creutzfeldt-Jakob disease, spongiform encephalopathy, light chain amyloidosis, Huntington’s disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado-Joseph disease, Alzheimer’s disease, bovine spongiform encephalopathy, scrapie, type 2 diabetes, amyloidosis caused by transthyretin (ATTR), AA type amyloidosis, Parkinson’s disease, Lewy body disease, traumatic brain injury, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis- related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, multiple myeloma, and frontotemporal dementia.

28. The method of claim 26 or 27, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, not including any insertions, to the amino acid sequence selected from the group selected from SEQ ID NO:13-18, 31-36, and 43-45, wherein the polypeptide includes plurality of alpha helical and beta strand domains, and wherein the polypeptide binds to a target peptide capable of forming a self-associating ȕ- strand.