Parallel Beta-sheet Structure Research Articles

Self-assembly of peptides: influence of substrate, pH and medium on the formation of supramolecular assemblies

Molecular self-assembly is a natural process occurring in many pathological conditions including Alzheimer's disease. Mechanistic insight into the self-assembly of amyloid fibrils led to the discovery of an aromatic dipeptide (diphenylalanine, FF) which is present in the core structural motif of Alzheimer's beta amyloid polypeptide. FF dipeptide has been reported to form well-defined nanotubes, which has been envisaged as a potential beta strand breaker in combination with charged amino acids for treating Alzheimer's disease. For therapeutic applications, it becomes important to consider the influence of hydrophobicity, temperature, pH, salts, proteins and peptide concentration on the self-assembly process which is the main focus of the present work. In the current study, we have qualitatively evaluated the morphological changes of FF nanostructures under various experimental conditions by scanning electron microscopy. Fourier Transform Infra-Red studies confirmed intermolecular hydrogen bonding and presence of parallel beta sheet structures. The scanning electron micrographs show formation of FF nanotubes under various conditions. The dimensions of the self-assembled nanostructures were found to be strongly influenced by the substrate, temperature, pH, salts and proteins. FF nanostructures formed under all experimental conditions showed enhanced fluorescence with Thioflavin T confirming the presence of beta sheet structures. The formation of peptide nanotubes in the physiological experimental conditions makes them attractive tool for future bionanotechnological applications.

In Situ Misfolding of Human Islet Amyloid Polypeptide at Interfaces Probed by Vibrational Sum Frequency Generation

Kinetic analysis of conformational changes of proteins at interfaces is crucial for understanding many biological processes at membrane surfaces. In this study, we demonstrate that surface-selective sum frequency generation (SFG) spectroscopy can be used to investigate kinetics of conformational changes of proteins at interfaces. We focus on an intrinsically disordered protein, human islet amyloid polypeptide (hIAPP) that is known to misfold into the beta-sheet structure upon interaction with membranes. Using the ssp polarization setting (s-polarized SFG, s-polarized visible, and p-polarized infrared), we observe changes in the amide I spectra of hIAPP at the air/water interface after addition of dipalmitoylphosphoglycerol (DPPG) that correspond to the lipid-induced changes in secondary structures. We also used the chiral-sensitive psp polarization setting to obtain amide I spectra and observed a gradual buildup of the chiral structures that display the vibrational characteristics of parallel beta-sheets. We speculate that the second-order chiral-optical response at the antisymmetric stretch frequency of parallel beta-sheet at 1622 cm(-1) could be a highly characteristic optical property of the beta-sheet aggregates not only for hIAPP, but possibly also for other amyloid proteins. Analyzing the achiral and chiral amide I spectra, we conclude that DPPG induces the misfolding of hIAPP from alpha-helical and random-coil structures to the parallel beta-sheet structure at the air/water interface. We propose that SFG could complement existing techniques in obtaining kinetic and structural information for probing structures and functions of proteins at interfaces.

Affinity-Based Screening of Peptides Recognizing Assembly States of Self-Assembling Peptide Nanomaterials

Novel peptides capable of binding to self-assembled peptide nanomaterials were identified from a random heptapeptide library displayed on phages. Affinity-dependent peptide screening against helically coiled nanofibers constructed of beta-sheet peptides gave phage clones displaying peptides with a variety of affinities and selectivities. An enzyme-linked immunosorbent assay using phage-displayed peptides revealed that the screened peptides specifically bind to target nanofibers, in contrast to reference nanofibers comprised of peptides with slightly different amino acid sequences. A Dot blot assay using chemically synthesized peptides demonstrated that peptide 01 (p01), with the sequence Thr-Gly-Val-Lys-Gly-Pro-Gly, showed an affinity constant (3.7 x 10(5) M(-1)) for the target nanofibers 200 times greater than its affinity for monomeric peptides and 60 times greater than for short nanofibers. These results suggested that p01 selectively recognizes the assembly state of the target peptide. ATR/IR secondary structure analyses clearly showed that when p01 binds to target nanofibers, it undergoes a structural transition from random-coil to parallel beta-sheet structures, resulting in greater affinity and high specificity for the target fiber. Surface modification of the peptide nanofibers by p01 demonstrated that the peptide specifically binds to the edge of the nanofibers. Using p01, uniform arrays of gold nanoparticles (proteins) could be generated on the peptide nanomaterials.

Evidence for novel beta-sheet structures in Iowa mutant beta-amyloid fibrils.

Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.

Macrocyclic Design Strategies for Small, Stable Parallel β-Sheet Scaffolds

Pairs of short peptide strands can be induced to adopt an antiparallel beta-sheet secondary structure in aqueous solution via a macrocyclic constraint, as illustrated by many natural and designed peptides. We show that an analogous strategy is successful for creation of small units of parallel beta-sheet secondary structure in aqueous solution. Cyclization in this case requires nonpeptide segments for N-to-N and C-to-C interstrand linkage. Surprisingly, we find that only one of these segments needs to be preorganized.

Nematic and Columnar Ordering of a PEG–Peptide Conjugate in Aqueous Solution

The self-assembly in aqueous solution of a PEG-peptide conjugate is studied by spectroscopy, electron microscopy, rheology and small-angle X-ray and neutron scattering (SAXS and SANS). The peptide fragment, FFKLVFF is based on fragment KLVFF of the amyloid beta-peptide, Abeta(16-20), extended by two hydrophobic phenylalanine units. This is conjugated to PEG which confers water solubility and leads to distinct self-assembled structures. Small-angle scattering reveals the formation of cylindrical fibrils comprising a peptide core and PEG corona. This constrained structure leads to a model parallel beta-sheet self-assembled structure with a radial arrangement of beta sheets. On increasing concentration, successively nematic and hexagonal columnar phases are formed. The flow-induced alignment of both structures was studied in situ by SANS using a Couette cell. Shear-induced alignment is responsible for the shear thinning behaviour observed by dynamic shear rheometry. Incomplete recovery of moduli after cessation of shear is consistent with the observation from SANS of retained orientation in the sample.

Characterization of β-Sheet Structure in Ure2p1-89 Yeast Prion Fibrils by Solid-State Nuclear Magnetic Resonance

Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole-dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N-13C and 13C-13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole-dipole couplings indicate that the beta-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel beta-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina [as originally reported in Siemer, A. B., Ritter, C., Ernst, M., Riek, R., and Meier, B. H. (2005) Angew. Chem., Int. Ed. 44, 2441-2444 and confirmed by measurements reported here] by factors of three or more, indicating a lower degree of structural order at the molecular level in Ure2p1-89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biological function of HET-s in heterokaryon incompatibility. Analysis of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid beta-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.

NMR Solution Structure of Subunit F of the Methanogenic A1AO Adenosine Triphosphate Synthase and Its Interaction with the Nucleotide-Binding Subunit B,

The A1AO adenosine triphosphate (ATP) synthase from archaea uses the ion gradients generated across the membrane sector (AO) to synthesize ATP in the A3B3 domain of the A1 sector. The energy coupling between the two active domains occurs via the so-called stalk part(s), to which the 12 kDa subunit F does belong. Here, we present the solution structure of the F subunit of the A1AO ATP synthase from Methanosarcina mazei Gö1. Subunit F exhibits a distinct two-domain structure, with the N-terminal having 78 residues and residues 79-101 forming the flexible C-terminal part. The well-ordered N-terminal domain is composed of a four-stranded parallel beta-sheet structure and three alpha-helices placed alternately. The two domains are loosely associated with more flexibility relative to each other. The flexibility of the C-terminal domain is further confirmed by dynamics studies. In addition, the affinity of binding of mutant subunit F, with a substitution of Trp100 against Tyr and Ile at the very C-terminal end, to the nucleotide-binding subunit B was determined quantitatively using the fluorescence signals of natural subunit B (Trp430). Finally, the arrangement of subunit F within the complex is presented.

Functional, Hierarchically Structured Poly(diacetylene)s via Supramolecular Self‐Assembly

The supramolecular self-assembly of macromonomers may serve as a first step to prepare well-defined, highly functionalized, hierarchically structured, conjugated polymers. Functional diacetylene macromonomers equipped with an oligopeptide segment designed to promote self-assembly into parallel beta-sheet type structures and a polydisperse, aliphatic coil segment to prevent global ordering give rise to supramolecular polymers with a tubular double-helical quarternary structure in organic solution. These supramolecular polymers may then be converted into the corresponding poly(diacetylene)s by UV irradiation under retention of their hierarchical structure.

NMR Reveals Anomalous Copper(II) Binding to the Amyloid Aβ Peptide of Alzheimer's Disease

The Abeta peptide is the major protein component of amyloid deposits in Alzheimer's disease (AD). Age-related microenvironmental changes in the AD brain promote amyloid formation that leads to cell injury and death. Altered levels of metals (such as Cu and Zn) exist in the AD brain, and because Cu and Zn can be bound to the Abeta in the amyloid plaques, it is thought that these binding events in vivo may trigger or prevent Abeta amyloid formation in the AD brain. Although several structural models have been proposed, all of these are undefined due to the lack of definitive structural data. The present NMR studies utilized uniformly 15N-labeled Abeta(1-40) peptide and 1H-15N HSQC experiments and demonstrate for the first time that the Abeta binds Cu and Zn in a distinct manner. The binding promotes NH signal disappearance of E3-V18, which was not due to the paramagnetic effect of Cu2+, as identical NMR studies were seen with Zn2+, which is diamagnetic. NMR titration experiments showed that the amide NH peak intensities of R5-L17 showed the most pronounced intensity reduction, and that the 1H signals for the side chain aromatic signals of the three histidines shift upfield (H6, H13, and H14). We propose that initially Cu2+ is anchored to the Abeta monomer (fast exchange rate) and is followed by deprotonation and/or severe line broadening of the backbone amide NH for E3-V18 (intermediate exchange rate). By contrast, Cu2+ binding to soluble Abeta aggregates leads to rapid aggregation and nonfibrillar amorphous structures, and without metal, the Abeta can undergo the normal time-dependent aggregation, eventually producing more ordered, late-stage parallel beta-sheet structures. These anomalous (rare) binding events may account for some of the unique properties associated with the Abeta, such as its proposed "dual role", where sequestration of metal ions by the monomer is neuroprotective, while that by beta-aggregates generates oxygen radicals and causes neuronal death.

Protein Denaturation and Aggregation

Protein aggregation is a prominent feature of many neurodegenerative diseases, such as Alzheimer's, Huntington's, and Parkinson's diseases, as well as spongiform encephalopathies and systemic amyloidoses. These diseases are sometimes called protein misfolding diseases, but the latter term begs the question of what is the "folded" state of proteins for which normal structure and function are unknown. Amyloid consists of linear, unbranched protein or peptide fibrils of approximately 100 A diameter. These fibrils are composed of a wide variety of proteins that have no sequence homology, and no similarity in three-dimensional structures--and yet, as fibrils, they share a common secondary structure, the beta-sheet. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidation of fibril structure. Recent advances in solid-state NMR spectroscopy and other biophysical techniques have led to the partial elucidation of fibril structure. Surprisingly at the time, for beta-amyloid, a set of 39-43-amino-acid peptides believed to play a pathogenic role in Alzheimer's disease, the beta-sheets are parallel with all amino acids of the sheets in-register. Since the time of those observations, however, it has become clear that there is no universal structure for amyloid fibrils. While many of the amyloid fibrils described thus far have a parallel beta-sheet structure, some have antiparallel beta-sheets, and other, more subtle structural differences among amyloids exist as well. Amyloids demonstrate conformational plasticity, the ability to adopt more than one stable tertiary fold. Conformational plasticity could account for "strain" differences in prions, and for the fact that a single polypeptide can form different fibril types with conformational differences at the atomic level. More recent data now indicate that the fibrils may not be the most potent or proximate mediators of cyto- and neurotoxicity. This damage is not confined to cell death, but also includes more subtle forms of damage, such as disruption of synaptic plasticity in the central nervous system. Rather than fibrils, prefibrillar aggregates, variously called "micelles," "protofibrils," or ADDLs (beta-amyloid-derived diffusible ligands in the case of beta-amyloid) may be the more proximate mediators of cell damage. These are soluble oligomers of aggregating peptides or proteins, but their structure is very challenging to study, because they are generally difficult to obtain in large enough quantities for high-resolution structural techniques, and they are temporally unstable, rapidly changing into more mature, and eventually fibrillar forms. Consequently, the mechanisms by which they disrupt cellular function are also not well understood. Nevertheless, three broad, overlapping, nonexclusive sets of mechanisms have been proposed as responsible for the cellular damage caused by soluble, oligomeric protein aggregates. These are: (1) disruption of cell membranes and their functions [e.g., by inserting into membranes and disrupting normal ion gradients]; (2) inactivation of normally folded, functional proteins [e.g., by sequestering or localizing transcription factors to the wrong cellular compartment]; and (3) "gumming up the works," by binding to and inactivating components of the quality-control system of cells, such as the proteasome or chaperone proteins.

Parallel beta-sheets and polar zippers in amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p.

We report the results of solid-state nuclear magnetic resonance (NMR) and atomic force microscopy measurements on amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p (Ure2p(10)(-)(39)). Measurements of intermolecular (13)C-(13)C nuclear magnetic dipole-dipole couplings indicate that Ure2p(10)(-)(39) fibrils contain in-register parallel beta-sheets. Measurements of intermolecular (15)N-(13)C dipole-dipole couplings, using a new solid-state NMR technique called DSQ-REDOR, are consistent with hydrogen bonds between side chain amide groups of Gln18 residues. Such side chain hydrogen bonding interactions have been called "polar zippers" by M. F. Perutz and have been proposed to stabilize amyloid fibrils formed by peptides with glutamine- and asparagine-rich sequences, such as Ure2p(10)(-)(39). We propose that polar zipper interactions account for the in-register parallel beta-sheet structure in Ure2p(10)(-)(39) fibrils and that similar peptides will also exhibit parallel beta-sheet structures in amyloid fibrils. We present molecular models for Ure2p(10)(-)(39) fibrils that are consistent with available experimental data. Finally, we show that solid-state (13)C NMR chemical shifts for (13)C-labeled Ure2p(10)(-)(39) fibrils are insensitive to hydration level, indicating that the fibril structure is not affected by the presence or absence of bulk water.

β-Sheet mediated self-assembly of dipeptides of ω-amino acids and remarkable fibrillation in the solid state

Single crystal X-ray diffraction studies show that the extended structure of dipeptide Boc-beta-Ala-m-ABA-OMe (m-ABA: meta-aminobenzoic acid) self-assembles in the solid state by intermolecular hydrogen bonding to create an infinite parallel beta-sheet structure. In dipeptide Boc-gamma-Abu-m-ABA-OMe (gamma-Abu: gamma-aminobutyric acid), two such parallel beta-sheets are further cross-linked by intermolecular hydrogen bonding through m-aminobenzoic acid moieties. SEM (scanning electron microscopy) studies reveal that both the peptides and form amyloid-like fibrils in the solid state. The fibrils are also found to be stained readily by Congo red, a characteristic feature of the amyloid fiber whose accumulation causes several fatal diseases such as Alzheimer's, prion-protein etc.

An Intersheet Packing Interaction in Aβ Fibrils Mapped by Disulfide Cross-Linking

Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.

Pathway complexity of Alzheimer's beta-amyloid Abeta16-22 peptide assembly.

Recent studies suggest that both soluble oligomers and insoluble fibrils have toxic effects in cell cultures, raising the interest in determining the first steps of the assembly process. We have determined the aggregation mechanisms of Abeta(16-22) dimer using the activation-relaxation technique and an approximate free energy model. Consistent with the NMR solid-state analysis, the dimer is predicted to prefer an antiparallel beta sheet structure with the expected registry of intermolecular hydrogen bonds. The simulations, however, locate three other antiparallel minima with nonnative beta sheet registries and one parallel beta sheet structure, slightly destabilized with respect to the ground state. This result is significant because it can explain the observed dependency of beta sheet registry on pH conditions. We also find that assembly of Abeta(16-22) into dimers follows multiple routes, but alpha-helical intermediates are not obligatory. This indicates that destabilization of alpha-helical intermediates is unlikely to abolish oligomerization of Abeta peptides.

Discrete molecular dynamics simulations of peptide aggregation.

We study the aggregation of peptides using the discrete molecular dynamics simulations. Specifically, at temperatures above the alpha-helix melting temperature of a single peptide, the model peptides aggregate into a multilayer parallel beta-sheet structure. This structure has an interstrand distance of 4.8 A and an intersheet distance of 10 A, which agree with experimental observations. Our model explains these results as follows: hydrogen-bond interactions give rise to the interstrand spacing in beta sheets, while Gō interactions between side chains make beta strands parallel to each other and allow beta sheets to pack into layers. An important feature of our results is that the aggregates contain free edges, which may allow for further aggregation of model peptides to form elongated fibrils.

Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study.

Structural and dynamical features of the B18 peptide from the sea urchin sperm binding protein were determined in the crystalline state and in zwitterionic lipid bilayers at a peptide:lipid molar ratio of 1:12 using solid-state NMR spectroscopy. The study was focused on three (13)C and (15)N uniformly labeled leucine residues, which were introduced into three different B18 peptides at positions evenly distributed along the B18 primary structure. Isotropic (13)C and (15)N chemical shift measurements showed that while B18 possesses a nonhelical and non-sheet-like structure in the crystalline state, the peptide adopts an oligomeric beta-sheet structure in the membrane in the presence of Zn(2+) ions at high peptide:lipid ratio. Torsion angle measurements for the three leucine sites supported these results, with phi torsion angles between -80 degrees and -90 degrees in the crystalline state and between -110 degrees and -120 degrees in the membrane-bound form. These phi torsion angles determined for membrane-bound B18 are consistent with a parallel beta-sheet secondary structure. Analysis of motionally averaged dipolar coupling measurements established an increase of the mobility in the leucine side chains upon binding to the membrane, whereas the backbone mobility remained essentially unchanged, except in the binding site of Zn(2+) ions. This difference in mobility was related to the H-bond network in the parallel beta-sheet structure, which involves the backbone and excludes the side chains of leucine residues. The parallel beta-sheet structure of B18 in the membrane in the presence of Zn(2+) appears to be an active state for the fusion of zwitterionic membranes in the presence of Zn(2+). A fluorescence fusion assay indicated that high B18 concentrations are required to induce fusion in these systems. Therefore, it was hypothesized that the oligomeric beta-sheet secondary structure revealed in the study represents an active state of the peptide in a membrane environment during fusion.

First crystallographic signature of amyloid-like fibril forming beta-sheet assemblage from a tripeptide with non-coded amino acids.

The model tripeptide Boc-beta-Ala(1)-Aib(2)-beta-Ala(3)-OMe 1 [beta-Ala: beta-alanine, Aib: alpha-aminoisobutyric acid] forms an infinite parallel beta-sheet structure through intermolecular interactions in single crystals and from the SEM diagram of this peptide, it is evident that the compound has fibrillar morphology, a characteristic of neurodegenerative disease causing amyloid aggregate.

Crystal structures of heterochiral peptides

Crystal structure of a heterochiral peptide, viz. Boc-Val1-D-Ala2-Leu3-Ala4-OMe with a D chiral residue in the second position of a sequence, has been determined [a = 40.44(1), b = 4.887(5), c = 15.381(5) A, beta = 109.6(1)degrees, space group C2, Z = 4, R = 0.11]. The peptide is in a parallel beta-sheet structure terminated by a distinct local bend. The structure is established by N-H...O as well as C alpha-H...O hydrogen bonds. The contiguous C alpha-H...O hydrogen bond observed in this structure is an unique observation.

Conformation of cyclo-bis(-L-valyl-L-prolyl-D-alanyl-), a synthetic cyclic hexapeptide

C26H42N6O6, Mr = 534.7, monoclinic, C2, a = 20.526 (2), b = 4.923 (1), c = 17.092 (2) A, beta = 126.37 (1) degrees, V = 1390.9 A3, Z = 2, Dm not measured, Dx = 1.28 g cm-3, lambda (Cu K alpha) = 1.5418 A, mu = 7.1 cm-1, F(000) = 576, R = 0.050, wR = 0.049 for 1012 reflections [I greater than 2 sigma (I)], 1501 unique reflections measured at room temperature (296 K). The synthetic cyclic hexapeptide, cyclo-bis(-L-Val-L-Pro-D-Ala-), exhibits exact C2 symmetry in the crystalline state with cis peptide links [omega = -13.1 (7) degrees] between Val and Pro residues; there are no intramolecular hydrogen bonds. The cyclic ring consists of two type VIb cis proline turns fused at the D-Ala residue. The backbone dihedral angles are all in the extended range except for psi Val [72.9 (5) degrees] and phi Pro [-78.9 (5) degrees] on either side of the cis peptide link. The carbonyl O atoms and the amide N atoms in the extended portion of the cyclic peptide form intermolecular hydrogen bonds with another cyclic hexapeptide molecule translated by a cell edge along the crystallographic b axis, forming an infinite stretch of beta-sheets. The parallel beta-sheet structures are separated by about 3.15 A.