Thermodynamic Selection of Steric Zipper Patterns in the Amyloid Cross-β Spine

At the core of amyloid fibrils is the cross-β spine, a long tape of β-sheets formed by the constituent proteins. Recent high-resolution x-ray studies show that the unit of this filamentous structure is a β-sheet bilayer with side chains within the bilayer forming a tightly interdigitating “steric zipper” interface. However, for a given peptide, different bilayer patterns are possible, and no quantitative explanation exists regarding which pattern is selected or under what condition there can be more than one pattern observed, exhibiting molecular polymorphism. We address the structural selection mechanism by performing molecular dynamics simulations to calculate the free energy of incorporating a peptide monomer into a β-sheet bilayer. We test filaments formed by several types of peptides including GNNQQNY, NNQQ, VEALYL, KLVFFAE and STVIIE, and find that the patterns with the lowest binding free energy correspond to available atomistic structures with high accuracy. Molecular polymorphism, as exhibited by NNQQ, is likely because there are more than one most stable structures whose binding free energies differ by less than the thermal energy. Detailed analysis of individual energy terms reveals that these short peptides are not strained nor do they lose much conformational entropy upon incorporating into a β-sheet bilayer. The selection of a bilayer pattern is determined mainly by the van der Waals and hydrophobic forces as a quantitative measure of shape complementarity among side chains between the β-sheets. The requirement for self-complementary steric zipper formation supports that amyloid fibrils form more easily among similar or same sequences, and it also makes parallel β-sheets generally preferred over anti-parallel ones. But the presence of charged side chains appears to kinetically drive anti-parallel β-sheets to form at early stages of assembly, after which the bilayer formation is likely driven by energetics.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Simulation Studies on the Stabilities of Aggregates Formed by Fibril-Forming Segments of α-Synuclein

Abstract

We performed molecular dynamics simulations for various oligomers with different β-sheet conformations consisting of α-Synuclein 71–82 residues using an all atom force field and explicit water model. Tetramers of antiparallel β-sheet are shown to be stable, whereas parallel sheets are highly unstable due to the repulsive interactions between bulky and polar side chains as well as the weaker backbone hydrogen bonds. We also investigated the stabilities of double antiparallel β-sheets stacked with asymmetric and symmetric geometries. Our results show that this 12 amino acid residue peptide can form stable β-sheet conformers at 320K and higher temperatures. The backbone hydrogen bonds in β-sheet and the steric packing between hydrophobic side chains between β-sheets are shown to give conformational stabilities.     

Increasing the Amphiphilicity of an Amyloidogenic Peptide Changes the β-Sheet Structure in the Fibrils from Antiparallel to Parallel

Solid-state NMR measurements have been reported for four peptides derived from β-amyloid peptide Aβ(1–42): Aβ(1–40), Aβ(10–35), Aβ(16–22), and Aβ(34–42). Of these, the first two are predicted to be amphiphilic and were reported to form parallel β-sheets, whereas the latter two peptides appear nonamphiphilic and adopt an antiparallel β-sheet organization. These results suggest that amphiphilicity may be significant in determining fibril structure. Here, we demonstrate that acylation of Aβ(16–22) with octanoic acid increases its amphiphilicity and changes the organization of fibrillar β-sheet from antiparallel to parallel. Electron microscopy, Congo Red binding, and one-dimensional ¹³C NMR measurements demonstrate that octanoyl-Aβ(16–22) forms typical amyloid fibrils. Based on the stability of monolayers at the air-water interface, octanoyl-Aβ(16–22) is more amphiphilic than Aβ(16–22). Measurements of ¹³C-¹³C and ¹⁵N-¹³C nuclear magnetic dipole-dipole couplings in isotopically labeled fibril samples, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) and rotational echo double resonance (REDOR) solid-state NMR techniques, demonstrate that octanoyl-Aβ(16–22) fibrils are composed of parallel β-sheets, whereas Aβ(16–22) fibrils are composed of antiparallel β-sheets. These data demonstrate that amphiphilicity is critical in determining the structural organization of β-sheets in the amyloid fibril. This work also shows that all amyloid fibrils do not share a common supramolecular structure, and suggests a method for controlling the structure of amyloid fibrils.     

β-Hairpin-Mediated Formation of Structurally Distinct Multimers of Neurotoxic Prion Peptides

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A₁₁₇V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies.     

Antiparallel Triple-strand Architecture for Prefibrillar Aβ42 Oligomers

Aβ42 oligomers play key roles in the pathogenesis of Alzheimer disease, but their structures remain elusive partly due to their transient nature. Here, we show that Aβ42 in a fusion construct can be trapped in a stable oligomer state, which recapitulates characteristics of prefibrillar Aβ42 oligomers and enables us to establish their detailed structures. Site-directed spin labeling and electron paramagnetic resonance studies provide structural restraints in terms of side chain mobility and intermolecular distances at all 42 residue positions. Using these restraints and other biophysical data, we present a novel atomic-level oligomer model. In our model, each Aβ42 protein forms a single β-sheet with three β-strands in an antiparallel arrangement. Each β-sheet consists of four Aβ42 molecules in a head-to-tail arrangement. Four β-sheets are packed together in a face-to-back fashion. The stacking of identical segments between different β-sheets within an oligomer suggests that prefibrillar oligomers may interconvert with fibrils via strand rotation, wherein β-strands undergo an ∼90° rotation along the strand direction. This work provides insights into rational design of therapeutics targeting the process of interconversion between toxic oligomers and non-toxic fibrils.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

Engineering Amyloid-Like Assemblies from Unstructured Peptides via Site-Specific Lipid Conjugation

Aggregation of amyloid beta (Aβ) into oligomers and fibrils is believed to play an important role in the development of Alzheimer’s disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of β-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1–2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of β-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.     

Investigating the effects of N-terminal acetylation on KFE8 self-assembly with 2D IR spectroscopy

Peptide self-assembly is an exciting and robust approach to create novel nanoscale materials for biomedical applications. However, the complex interplay between intra- and intermolecular interactions in peptide aggregation means that minor changes in peptide sequence can yield dramatic changes in supramolecular structure. Here, we use two-dimensional infrared spectroscopy to study a model amphiphilic peptide, KFE8, and its N-terminal acetylated counterpart, AcKFE8. Two-dimensional infrared spectra of isotope-labeled peptides reveal that AcKFE8 aggregates comprise two distinct β-sheet structures although KFE8 aggregates comprise only one of these structures. Using an excitonic Hamiltonian to simulate the vibrational spectra of model β-sheets, we determine that the spectra are consistent with antiparallel β-sheets with different strand alignments, specifically a two-residue shift in the register of the β-strands. These findings bring forth new insights into how N-terminal acetylation may subtly impact secondary structure, leading to larger effects on overall aggregate morphology. In addition, these results highlight the importance of understanding the residue-level structural differences that result from changes in peptide sequence to facilitate the rational design of peptide materials.     

Human cerebral vascular amyloid contains both antiparallel and parallel in-register Aβ40 fibrils

The accumulation of fibrillar amyloid-β (Aβ) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer''s disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aβ mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aβ fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel β-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aβ fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aβ fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aβ40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel β-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register β-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aβ fibrils in the human brain.     

Formation and Growth of Oligomers: A Monte Carlo Study of an Amyloid Tau Fragment

Small oligomers formed early in the process of amyloid fibril formation may be the major toxic species in Alzheimer's disease. We investigate the early stages of amyloid aggregation for the tau fragment AcPHF6 (Ac-VQIVYK-NH2) using an implicit solvent all-atom model and extensive Monte Carlo simulations of 12, 24, and 36 chains. A variety of small metastable aggregates form and dissolve until an aggregate of a critical size and conformation arises. However, the stable oligomers, which are β-sheet-rich and feature many hydrophobic contacts, are not always growth-ready. The simulations indicate instead that these supercritical oligomers spend a lengthy period in equilibrium in which considerable reorganization takes place accompanied by exchange of chains with the solution. Growth competence of the stable oligomers correlates with the alignment of the strands in the β-sheets. The larger aggregates seen in our simulations are all composed of two twisted β-sheets, packed against each other with hydrophobic side chains at the sheet–sheet interface. These β-sandwiches show similarities with the proposed steric zipper structure for PHF6 fibrils but have a mixed parallel/antiparallel β-strand organization as opposed to the parallel organization found in experiments on fibrils. Interestingly, we find that the fraction of parallel β-sheet structure increases with aggregate size. We speculate that the reorganization of the β-sheets into parallel ones is an important rate-limiting step in the formation of PHF6 fibrils.     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.     

Molecular dynamics simulation of the nanofibrils formed by amyloid-based peptide amphiphiles

Abstract

Atomistic molecular dynamics simulations have been performed on the peptide amphiphiles (PAs) with four amyloid beta peptide fragments as head groups. The stable structures were monitored by the root mean square deviation with respect to the energy minimised initial structures. Random coil and β-sheet structures with hydrogen bonds along and perpendicular to the long axis of the nanofibre were obtained due to the different nature of the head groups. Influences of pH and capping ends on the nanofibre structures were investigated through variation of the protonation states of the ionic amino acids in the peptides. The peptides with opposite charges on both sides were found to have the fewest β-sheet structures, and the charges on the outer terminal tended to destruct the β-sheets while those at the inner side did not. The isolated charge in the centre of peptides was found to be able to promote the formation of regular β-sheets, while multiple charged residues could not support ordered β-sheet structures. When charge neutralisation occurred between adjacent residues, regular β-sheet laminates might also occur for systems with charges at the outer terminal. With the increase of β-sheet structures formed, the original twisted structures found for random coil structures of the PAs could be diminished by the hydrogen bonds.     

Molecular Self-Assembly Strategy for Generating Catalytic Hybrid Polypeptides

Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.     

Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work.     

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ_1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ_1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH₂ and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH₂) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH₂). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ_1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH₂ β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ_1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ_1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ_1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH₂ β-sheet breaker.     

Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)₃GY(GA)₃GE(GA)₃GH(GA)₃GK, forms large β-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of β-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel β-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125°C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of ~1 and ~60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal “nonnative” interactions. The polypeptide folding dynamics agree with a general property of β-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.     

Structural investigations of lipid, polypeptide and protein multilayers

Langmuir-Blodgett multilayers of lipids, polypeptides and proteins have been examined by X-ray diffraction and infrared spectroscopic methods. The complex polymorphism exhibited by multilayers of glycerides and various phospholipids of different chain length mirror those shown in other three-dimensional structures and suggest that multilayers of lipids can be considered as oriented “crystals”. Both the α and β types of hdyrocarbon chain packing are adopted by different classes of lipids in multilayers.Stable multilayers of the synthetic polypeptide poly-γ-benzyl-l-glutamate consist of α-helical rods stacked in an hexagonal array with a rod axis separation of 14.2 Å. Poly-γ-methyl-l-glutamate behaves similarly but little structural information could be derived from potentially non-helical or sheet-like structures formed by other homopolypeptides. The observation of a single, invariant diffraction line at 9.3 Å for multilayers of a number of water-soluble proteins is consistent with the occurrence of extensive structural reorganization (uncoiling, denaturation) at the air-water interface.