Hybrid peptides: expanding the beta turn in peptide hairpins by the insertion of beta-, gamma-, and delta-residues

The beta turn segment in designed peptide hairpins has been expanded by the insertion of beta-, gamma- and delta-amino acids at the i+2 position. The model octapeptides Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe (1), Boc-Leu-Phe-Val-DPro-beta3-Ac6c-Leu-Phe-Val-OMe (2), and Boc-Leu-Phe-Val-DPro-Gpn-Leu-Phe-Val-OMe (3) have been shown to adopt beta hairpin conformations in methanol by the observation of key diagnostic nuclear Overhauser effects. Boc-Leu-Val-Val-DPro-delta-Ava-Leu-Val-Val-OMe (4) adopts a beta hairpin conformation in crystals; this is stabilized by three cross-strand hydrogen bonds as demonstrated by X-ray diffraction. The canonical C10 turn in an alpha-alpha segment is expanded to C11, C12, and C13 turns in alpha-beta, alpha-gamma, and alpha-delta segments, respectively. The crystal structures of Piv-LPro-beta3-Ac6c-NHMe (5) and Boc-Ac6c-Gpn-Ac6c-OMe (6) reveal intramolecularly hydrogen-bonded C11 and C12 conformations, respectively. Computer modeling of octapeptide sequences that contain centrally positioned hybrid-turn segments, by using turn parameters derived from the structures of peptides 5 and 6, establishes the stereochemical acceptability of the beta hairpins in the cases of peptides 2 and 3. Accommodation of omega-amino acids into the turn segments is achieved by the adoption of gauche conformations around the backbone C--C bonds.     

Hybrid peptide hairpins containing alpha- and omega-amino acids: conformational analysis of decapeptides with unsubstituted beta-, gamma-, and delta-residues at positions 3 and 8

The effects of inserting unsubstituted omega-amino acids into the strand segments of model beta-hairpin peptides was investigated by using four synthetic decapeptides, Boc-Leu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val-OMe: peptide 1 (Xxx=Gly), peptide 2 (Xxx=betaGly=betahGly=homoglycine, beta-glycine), peptide 3 (Xxx=gammaAbu=gamma-aminobutyric acid), peptide 4 (Xxx=deltaAva=delta-aminovaleric acid). 1H NMR studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for beta-hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a beta-hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the gammaAbu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II' beta-turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 nm, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.     

Expected and unexpected results from combined beta-hairpin design elements

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.     

Stability of cyclic beta-hairpins: asymmetric contributions from side chains of a hydrogen-bonded cross-strand residue pair

Amino acid structural propensities measured in "host-guest" model studies are often used in protein structure prediction or to choose appropriate residues in de novo protein design. While this concept has proven useful for helical structures, it is more difficult to apply successfully to beta-sheets. We have developed a cyclic beta-hairpin scaffold as a host for measurement of individual residue contributions to hairpin structural stability. Previously, we have characterized substitutions in non-backbone-hydrogen-bonded strand sites; relative stability differences measured in the cyclic host are highly predictive of changes in folding free energy for linear beta-hairpin peptides. Here, we examine the hydrogen-bonded strand positions of our host. Surprisingly, we find a large favorable contribution to stability from a valine (or isoleucine) substitution immediately preceding the C-terminal cysteine of the host peptide, but not at the cross-strand position of the host or in either strand of a folded linear beta-hairpin (trpzip peptide). Further substitutions in the peptides and NMR structural analysis indicate that the stabilizing effect of valine is general for CX(8)C cyclic hairpins and cannot be explained by particular side-chain-side-chain interactions. Instead, a localized decrease in twist of the peptide backbone on the N-terminal side of the cysteine allows the valine side chain to adopt a unique conformation that decreases the solvent accessibility of the peptide backbone. The conformation differs from the highly twisted (coiled) conformation of the trpzip hairpins and is more typical of conformations present in multistranded beta-sheets. This unexpected structural fine-tuning may explain why cyclic hairpins selected from phage-displayed libraries often have valine in the same position, preceding the C-terminal cysteine. It also emphasizes the diversity of structures accessible to beta-strands and the importance of considering not only "beta-propensity", but also hydrogen-bonding pattern and strand twist, when designing beta structures. Finally, we observe correlated, cooperative stabilization from side-chain substitutions on opposite faces of the hairpin. This suggests that cooperative folding in beta-hairpins and other small beta-structures is driven by cooperative strand-strand association.     

Molecular dynamics study of peptides in implicit water: ab initio folding of beta-hairpin, beta-sheet, and beta beta alpha-motif

In this communication, we have demonstrated that molecular dynamics simulations using a GB implicit solvation model with the all-atom based force field (CHARMM19) can describe the spontaneous folding of small peptides in aqueous solution. The native structures of peptides with various structural motifs (beta-hairpin, beta-sheet, and betabetaalpha-moiety) were successfully predicted within reasonable time scales by MD simulations at moderately elevated temperatures. It is expected that the present simulations provide further insight into mechanism/pathways of the peptide folding.     

Intermolecular packing and alignment in an ordered beta-hairpin antimicrobial peptide aggregate from 2D solid-state NMR

The aggregation and packing of a membrane-disruptive beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), in the solid state are investigated to understand its oligomerization and hydrogen-bonding propensity. Incubation of PG-1 in phosphate buffer saline produced well-ordered nanometer-scale aggregates, as indicated by 13C and 15N NMR line widths, chemical shifts, and electron microscopy. Two-dimensional 13C and 1H spin diffusion experiments using C-terminus strand and N-terminus strand labeled peptides indicate that the beta-hairpin molecules in these ordered aggregates are oriented parallel to each other with like strands lining the intermolecular interface. In comparison, disordered and lyophilized peptide samples are randomly packed with both parallel and antiparallel alignments. The PG-1 aggregates show significant immobilization of the Phe ring near the beta-turn, further supporting the structural ordering. The intermolecular packing of PG-1 found in the solid state is consistent with its oligomerization in lipid bilayers. This solid-state aggregation approach may be useful for determining the quaternary structure of peptides in general and for gaining insights into the oligomerization of antimicrobial peptides in lipid bilayers in particular.     

Thermodynamic analysis of beta-hairpin-forming peptides from the thermal dependence of (1)H NMR chemical shifts

The temperature dependence of the (1)H chemical shifts of six designed peptides previously shown to adopt beta-hairpin structures in aqueous solution has been analyzed in terms of two-state (beta-hairpin left arrow over right arrow coil) equilibrium. The stability of the beta-hairpins formed by these peptides, as derived from their T(m) (midpoint transition temperature) values, parallels in general their ability to adopt those structures as deduced from independent NMR parameters: NOEs, Deltadelta(C)(alpha)(H), Deltadelta(C)(alpha), and Deltadelta(C)(beta) values. The observed T(m) values are dependent on the particular position within the beta-hairpin that is probed, indicating that their folding to a beta-hairpin conformation deviates from a "true" two-state transition. To obtain individual T(m) values for each hairpin region in each peptide, a simplified model of a successive uncoupled two-state equilibrium covering the entire process has been applied. The distribution of T(m) values obtained for the different beta-hairpin regions (turn, strands, backbone, side chains) in the six analyzed peptides reveals a similar pattern. A model for beta-hairpin folding is proposed on the basis of this pattern and the reasonable assumption that regions showing higher T(m) values are the last ones to unfold and, presumably, the first to form. With this assumption, the analysis suggests that turn formation is the first event in beta-hairpin folding. This is consistent with previous results on the essential role of the turn sequence in beta-hairpin folding.     

β‐Hairpins Generated from Hybrid Peptide Sequences Containing both α‐ and β‐Amino Acids

The incorporation of the β‐amino acid residues into specific positions in the strands and β‐turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C(α) C(β) bond (θ) enhances the conformational repertoire in β‐residues. The conformational analysis of three designed peptide hairpins composed of α/β‐hybrid segments is described: Boc‐Leu‐Val‐Val‐^DPro‐β Phe ‐Leu‐Val‐Val‐OMe ( 1 ), Boc‐Leu‐Val‐β Val ‐^DPro‐Gly‐β Leu ‐Val‐Val‐OMe ( 2 ), and Boc‐Leu‐Val‐β Phe ‐Val‐^DPro‐Gly‐Leu‐β Phe ‐Val‐Val‐OMe ( 3 ). 500‐MHz ¹H‐NMR Analysis supports a preponderance of β‐hairpin conformation in solution for all three peptides, with critical cross‐strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a β‐hairpin conformation with two β‐residues occupying facing, non‐H‐bonded positions in antiparallel β‐strands. Notably, βVal(3) adopts a gauche conformation about the C(α) C(β) bond (θ=+65°) without disturbing cross‐strand H‐bonding. The crystal structure of 2 , together with previously published crystal structures of peptides 3 and Boc‐β Phe ‐β Phe ‐^DPro‐Gly‐β Phe ‐β Phe ‐OMe, provide an opportunity to visualize the packing of peptide sheets with local ‘polar segments' formed as a consequence of reversal peptide‐bond orientation. The available structural evidence for hairpins suggests that β‐residues can be accommodated into nucleating turn segments and into both the H‐bonding and non‐H‐bonding positions on the strands.     

Adsorption of amyloid beta (1-40) peptide to phosphatidylethanolamine monolayers.

The aggregation of soluble, nontoxic amyloid beta (Abeta) peptide to beta-sheet containing fibrils is assumed to be a major step in the development of Alzheimer's disease. Interactions of Abeta with neuronal membranes could play a key role in the pathogenesis of the disease. Herein, we study the adsorption of synthetic Abeta peptide to DPPE and DMPE monolayers (dipalmitoyl- and dimyristoylphosphatidylethanolamine). Both lipids exhibit a condensed monolayer state at 20 degrees C and form a similar lattice. However, at low packing densities (at large area per molecule), the length of the acyl chains determines the phase behavior, therefore DPPE is fully condensed whereas DMPE exhibits a liquid-expanded state with a phase transition at approximately 5-6 mNm(-1). Adsorption of Abeta to DPPE and DMPE monolayers at low surface pressure leads to an increase of the surface pressure to approximately 17 mNm(-1). The same was observed during adsorption of the peptide to a pure air-water interface. Grazing incidence X-ray diffraction (GIXD) experiments show no influence of Abeta on the lipid structure. The adsorption kinetics of Abeta to a DMPE monolayer followed by IRRAS (infrared reflection absorption spectroscopy) reveals the phase transition of DMPE molecules from liquid-expanded to condensed states at the same surface pressure as for DMPE on pure water. These facts indicate no specific interactions of the peptide with either lipid. In addition, no adsorption or penetration of the peptide into the lipid monolayers was observed at surface pressures above 30 mNm(-1). IRRAS allows the measurement of the conformation and orientation of the peptide adsorbed to the air-water interface and to a lipid monolayer. In both cases, with lipids at surface pressures below 20 mNm(-1) and at the air-water interface, adsorbed Abeta has a beta-sheet conformation and these beta-sheets are oriented parallel to the interface.     

NMR analysis of aromatic interactions in designed peptide beta-hairpins

Designed octapeptide beta-hairpins containing a central (D)Pro-Gly segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel beta-strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel beta-sheets, in the non-hydrogen bonding position (Ac-L-Y-V-(D)P-G-L-Y/W-V-OMe: peptides 1 and 2), diagonally across the strands (Boc-Y/W-L-V-(D)P-G-W-L-V-OMe: peptides 3 and 6), and along the strands at positions i and i + 2 (Boc-L-L-V-(D)P-G-Y-L-W-OMe: peptide 4). Two peptides served as controls (Boc-L-L-V-(D)P-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-(D)P-G-L-L-V-OMe: peptide 7) for aromatic interactions. All studies have been carried out using solution NMR methods in CDCl(3) + 10% DMSO-d(6) and have been additionally examined in CD(3)OH for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces.     

Fabrication of nanofibers with uniform morphology by self-assembly of designed peptides

Fabrication of controlled peptide nanofibers with homogeneous morphology has been demonstrated. Amphiphilic beta-sheet peptides were designed as sequences of Pro-Lys-X(1)-Lys-X(2)-X(2)-Glu-X(1)-Glu-Pro. X(1) and X(2) were hydrophobic residues selected from Phe, Ile, Val, or Tyr. The peptide FI (X(1)=Phe; X(2)=Ile) self-assemble into straight fibers with 80-120 nm widths and clear edges, as examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The fiber formation is performed in a hierarchical manner: beta-sheet peptides form a protofibril, the protofibrils assemble side-by-side to form a ribbon, and the ribbons then coil in a left-handed fashion to make up a straight fiber. These type of fibers are formed from peptides possessing hydrophobic aromatic Phe residue(s). Furthermore, a peptide with Ala residues at both N and C termini does not form fibers (100 nm scale) with clear edges; this causes random aggregation of small pieces of fibers instead. Thus, the combination of unique amphiphilic sequences and terminal Pro residues determine the fiber morphology.     

Aggregation of polyalanine in a hydrophobic environment

The dimerization of polyalanine peptides in a hydrophobic environment was explored using replica exchange molecular dynamics simulations. A nonpolar solvent (cyclohexane) was used to mimic, among other hydrophobic environments, the hydrophobic interior of a membrane in which the peptides are fully embedded. Our simulations reveal that while the polyalanine monomer preferentially adopts a beta-hairpin conformation, dimeric phases exist in an equilibrium between random coil, alpha-helical, beta-sheet, and beta-hairpin states. A thermodynamic characterization of the dimeric phases reveals that electric dipole-dipole interactions and optimal side-chain packing stabilize alpha-helical conformations, while hydrogen bond interactions favor beta-sheet conformations. Possible pathways leading to the formation of alpha-helical and beta-sheet dimers are discussed.     

Folding mechanisms of individual beta-hairpins in a Go model of Pin1 WW domain by all-atom molecular dynamics simulations

This paper examines the folding mechanism of an individual beta-hairpin in the presence of other hairpins by using an off-lattice model of a small triple-stranded antiparallel beta-sheet protein, Pin1 WW domain. The turn zipper model and the hydrophobic collapse model originally developed for a single beta-hairpin in literature is confirmed to be useful in describing beta-hairpins in model Pin1 WW domain. We find that the mechanism for folding a specific hairpin is independent of whether it folds first or second, but the formation process are significantly dependent on temperature. More specifically, beta1-beta2 hairpin folds via the turn zipper model at a low temperature and the hydrophobic collapse model at a high temperature, while the folding of beta2-beta3 hairpin follows the turn zipper model at both temperatures. The change in folding mechanisms is interpreted by the interplay between contact stability (enthalpy) and loop lengths (entropy), the effect of which is temperature dependent.     

Nucleation of beta-hairpin structures with cis amide bonds in E-vinylogous proline-containing peptides

Synthesis and conformational studies of peptides containing the E-vinylogous prolines 1 (VPro1) and 2 (VPro2), Boc-Ala-Val-VPro1-Xaa-Leu-OMe (3, Xaa = Gly; 4, Xaa = Phe), Boc-Ala-Val-VPro2-Xaa-Leu-OMe (5, Xaa = Gly; 6, Xaa = Phe), Boc-Leu-Ile-Val-VPro1-Xaa-Leu-OMe (7, Xaa = Gly; 8, Xaa = Phe), and Boc-Leu-Ile-Val-VPro2-Xaa-Leu-OMe (9, Xaa = Gly; 10, Xaa = Phe), were carried out. It has been shown that both VPro1 and VPro2 lead to the formation of 12-membered intramolecularly hydrogen bonded structures very similar to type VI beta-turns with a cis Xaa-VPro amide bond in the major conformers in all the peptides 3-10, resulting in the nucleation of beta-hairpin type structures in these molecules in CDCl(3).     

Determination of peptide oligomerization in lipid bilayers using 19F spin diffusion NMR

Aggregation or oligomerization is important for the function of many membrane peptides such as ion channels and antimicrobial peptides. However, direct proof of aggregation and the determination of the number of molecules in the aggregate have been difficult due to the lack of suitable high-resolution methods for membrane peptides. We propose a 19F spin diffusion magic-angle-spinning NMR technique to determine the oligomeric state of peptides bound to the lipid bilayer. Magnetization transfer between chemically equivalent but orientationally different 19F spins on different molecules reduces the 19F magnetization in an exchange experiment. At long mixing times, the equilibrium 19F magnetization is 1/M, where M is the number of orientationally different molecules in the aggregate. The use of the 19F spin increases the homonuclear dipolar coupling and thus the distance reach. We demonstrate this technique on crystalline model compounds with known numbers of molecules in the asymmetric unit cell, and show that 19F spin diffusion is more efficient than that of 13C by a factor of approximately 500. Application to a beta-hairpin antimicrobial peptide, protegrin-1, shows that the peptide is almost completely dimerized in POPC bilayers at a concentration of 7.4 mol %. Decreasing the peptide concentration reduced the dimer fraction. Using a monomer-dimer equilibrium model, we estimate the DeltaG for dimer formation to be -10.2 +/- 2.3 kJ/mol. This is in good agreement with the previously measured free energy reduction for partitioning and aggregating beta-sheet peptides into phospholipid membranes. This 19F spin diffusion technique opens the possibility of determining the oligomeric structures of membrane peptides.     

Ion mobility-mass spectrometry applied to cyclic peptide analysis: Conformational preferences of gramicidin S and linear analogs in the gas phase

In this paper, we present an investigation of the gas-phase structural differences between cyclic and linear peptide ions by matrix-assisted laser desorption ionization-ion mobility-mass spectrometry. Specifically, data is shown for gramicidin S (cyclo-VOLFPVOLFP where phenylalanines are D rather than L-type amino acids and the O designates the non-standard amino acid ornithine) and five linear gramicidin S analogues. Results are interpreted as evidence for a beta-sheet (or beta-hairpin) conformational preference in both linear-protonated and sodiated-cyclic gramicidin S gas-phase peptides, and a preference for the protonated-cyclic peptide to adopt a collapsed, random coil-type conformation. A comparison with solution-phase circular dichroism measurements is performed, and structures similar to those observed in the gas phase appear to be favored in low-dielectric solvents such as 2,2,2-triflouroethanol. The utility of ion mobility-mass spectrometry (IM-MS) as a means of rapidly distinguishing between linear and cyclic peptide forms in also discussed.     

Beta-hairpin folding by a model amyloid peptide in solution and at an interface

The development of specific agents against amyloidoses requires an understanding of the conformational distribution of fibrillogenic peptides at a microscopic level. Here, I present molecular dynamics simulations of the model amyloid peptide LSFD with sequence LSFDNSGAITIG-NH2 in explicit water and at a water/vapor interface for a total time scale of approximately 1.8 micros. An extended structure was used as initial peptide configuration. At approximately 290 K, solvated LSFD was kinetically trapped in diverse misfolded beta-sheet/coil conformations. At 350 K, in contrast, the same type II' beta-hairpin in equilibrium with less ordered but also U-shaped conformations was observed for the core residues DNSGAITI in solution and at the interface in multiple independent simulations. The most stable structural unit of the beta-hairpin was the two residue turn (GA). The core residues exhibited a well-defined folded state in which the beta-hairpin was stabilized by a hydrogen bond between the side chain of Asn-385 and the main chain carbonyl group of Gly-387. My results suggest that beta-sheet conformations indicated from previous Fourier-transform infrared spectroscopy measurements immediately after preparation of the peptide solution may not arise from protofilaments as speculated by others but are a property of LSFD monomers. In addition, combined with previous results from X-ray scattering, my findings suggest that interfacial aggregation of LSFD implies a transition from U-shaped to extended peptide conformations. This work including the first simulations of reversible beta-hairpin folding at an interface is an essential step toward a microscopic understanding of interfacial peptide folding and self-assembly. Knowledge of the main conformation of the peptide core may facilitate the design of possible inhibitors of LSFD aggregation as a test ground for future computational therapeutic strategies against amyloid diseases.     

Solution NMR studies of the A beta(1-40) and A beta(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A beta(1-40) and 42-residue A beta(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A beta peptide building blocks into multimeric fibrillar beta-strand aggregates remain unknown. Recently, we established that oxidation of the methionine-35 side chain to the sulfoxide (Met35(red) --> Met35(ox)) significantly impedes the rate of aggregation and fibrillation of the A beta peptide. To explore this effect at greater resolution, we carefully compared the (1)H, (15)N, and (13)C NMR chemical shifts of four A beta peptides that had the Met35 reduced or oxidized (A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox)). With the use of a special disaggregation protocol, the highly aggregation prone A beta peptides could be studied at higher, millimolar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5 degrees C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the (1)H alpha, (13)C alpha, and (13)C beta established that the four peptides are largely random, extended chain structures, with the Met35(ox) reducing the propensity for beta-strand structure at two hydrophobic regions (Leu17-Ala21 and Ile31-Val36), and turn- or bendlike structures at Asp7-Glu11 and Phe20-Ser26. Additional NMR studies monitoring changes that occur during aging at 37 degrees C established that, along with a gradual loss of signal/noise, the Met35(ox) significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, His13, and His14 side chains. Taken together, the present NMR studies demonstrate that the Met35(red) --> Met35(ox) conversion prevents aggregation by reducing both hydrophobic and electrostatic association and that the A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox) peptides may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.     

Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as silk I and II

The influence of the bulky and H-bonding Tyr side-chain on its Ala- and Gly-rich environment in Bombyx mori silk fibroin was examined by (13)C cross-polarization magic angle spinning (CP/MAS), static (2)H and (19)F NMR and molecular mechanics calculations. Model peptides of the type (AG)(15) were synthesized with Tyr in a number of different positions, precipitated under conditions favoring either of the two characteristic protein conformations, and the resulting structures were assigned from their (13)C chemical shifts. Dialysis of native fibroin or the simple (AG)(15) peptide from a 9 M LiBr solution against water produces silk I (the structure of silk before spinning), whereas drying from formic acid yields silk II (fibrous structure after spinning). We found that the introduction one or more Tyr into (AG)(15) can have a dramatic effect not only on the local backbone conformation but also on the long-range intermolecular chain packing in the samples. The antiparallel beta-sheet conformation of silk II is able readily to accommodate a single Tyr residue. Interestingly, the beta-turn conformation of silk I only remains stable when Tyr is positioned near the chain terminus in (AG)(12)YG(AG)(2), but the conformation is driven towards silk II when Tyr is located in the central region of (AG)(7)YG(AG)(7). The role of H-bonding was tested by replacing Tyr with Phe or 4F-Phe, which are no longer compatible with silk I and fully induced a silk II conformation. In the presence of several Tyr residues a mixture of distorted beta-sheet and beta-turn conformations was obtained, regardless of the precipitation conditions. Static (2)H NMR of ring-deuterated [3',5'-(2)H(2)]Tyr located in the central region of (AG)(7)YG(AG)(7) showed that the side-chain is immobilized in both silk I and II, which was also observed by static (19)F NMR of the 4F-Phe analogue. To visualize the local packing around the Tyr side-chain, molecular mechanics calculations were performed on a mixture of (AG)(4) and AGAGYGAG, starting from either the beta-turn type II or the antiparallel beta-sheet structure. The resulting structures show that the intermolecular chain arrangement is significantly affected by Tyr, thus explaining the long-range packing effects in the semi-crystalline regions of silk fibers compared with the crystalline regions that are devoid of Tyr.     

Conformation-dependent racemization of aspartyl residues in peptides

Biologically uncommon D-aspartyl (D-Asp) residues have been detected in proteins of various tissues of elderly humans. The presence of D-Asp has been explained as a result of the racemization of L-Asp (denoted as Asp) in the protein of inert tissues. We have previously suggested that the racemization of Asp may depend on the conformation of the peptide chain. However, the nature of the peptide conformation that affects the D-Asp formation has not yet been examined. Here we report the kinetics of Asp racemization in two model peptides, (Asp-Leu)(15) and (Leu-Asp-Asp-Leu)(8)-Asp, which form beta-sheet structures and alpha-helical structures, respectively. For the beta-sheet structures, the activation energy of racemization of Asp residues was 27.3 kcal mol(-1), the racemization rate constant at 37 degrees C was 2.14x10(-2) per year and the time required to reach a D/L ratio of 0.99 at 37 degrees C was 122.6 years as estimated from the Arrhenius equation. For the alpha-helical structures, the activation energy of racemization was 18.4 kcal mol(-1), the racemization rate constant 20.02x10(-2) per year and the time 13.1 year. These results suggest that Asp residues inserted in alpha-helical peptides are more sensitive to racemization than Asp residues inserted in peptides adopting beta-sheet structures. The results clearly indicate that the racemization rate of Asp residues in peptides depends on the secondary structure of the host peptide.