Conformational diversity of the fibrillogenic fusion peptide B18 in different environments from molecular dynamics simulations

The development of specific agents against amyloidoses requires an understanding of the conformational behavior of fibrillogenic peptides in different environments on the microscopic level. We present extensive molecular dynamics simulations of the fibrillogenic Bindin (103-120) B18 fusion peptide for several different environments: a water-trifluorethanol (TFE) mixture, pure water, aqueous buffer containing 100 mM NaCl, and a buffer-vapor interface. The peptide was studied as an isolated molecule in solution or at an interface. In the simulations, the conformational behavior of the peptide was found to strongly depend on the environment in agreement with experimental data. Overall, large portions of the peptide were unstructured. Preformed alpha-helical conformations were least stable in pure water and most stable in the water-TFE mixture and the buffer-vapor interface. In all environments, the alpha-helical conformation was most stable in the region around residues 113-116, which are mainly hydrophilic. Extended configurations in water or buffer folded into structures containing beta-sheets in agreement with data from circular dichroism spectroscopy. In buffer, the beta-sheet content was larger than in water and alpha-beta transitions were observed at elevated temperature. Beta-sheets were formed by hydrophobic residues; turns were formed by hydrophilic residues. A few typical beta-sheets that contain different residues are suggested. A B18 molecule in a strand-loop-strand conformation placed in buffer in contact with vapor was spontaneously adsorbed to the buffer-vapor interface with its hydrophobic side pointing toward the vapor phase. The adsorption induced the formation of turns at positions 108-119 and alpha-helical conformations in the region around residues 114-117. Alpha-helices were parallel to the interface plane in agreement with data from IR reflection absorption spectroscopy.     

Evaluation of peptide-peptide interactions using reversed-phase high-performance liquid chromatography.

The separation of peptides during RP-HPLC depends mainly upon differential hydrophobic interactions of the individual peptides being separated with the C18 group of the stationary phase. We have examined the behavior of dimeric disulfide-linked model peptides during RP-HPLC in order to study self-induced conformational effects. A set of 18 analogues of the amphipathic alpha-helical sequence Ac-LKLLKKLLKKLKKLLKKL-NH2 was used for this study. These analogues differed only by the successive replacement of each position with a cysteine. Strong peptide-peptide interactions, occurring through interchain hydrophobic forces, resulted in a presenting face to the C18 group, consisting primarily of lysine residues and, in turn, in early retention times. Three homo-dimers were also found to be strongly alpha-helical in water as determined by circular dichroism spectroscopy.     

Comparison of the conformational stability of the non-native alpha-helical intermediate of thiol-modified beta-lactoglobulin upon interaction with sodium n-alkyl sulfates at two different pH

Bovine beta-lactoglobulin assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. beta-lactoglobulin has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major or alpha-helix. This thiol group was specifically reacted with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) at pH 7.5 and 2, producing a modified beta-lactoglobulin containing a mix disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of thiol modified beta-lactoglobulin (TNB-beta-LG) was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at pH 7.5 and 2. The conformation and stability of non-native alpha-helical intermediate (alphaI) state of TNB-beta-LG were studied by circular dichroism (CD), fluorescence and differential scanning calorimetry (DSC) techniques. The effect of n-alkyl sulfates on the structure of alphaI state at both pH was utilized to investigate the contribution of hydrophobic interactions to the stability of alphaI intermediate. The present results suggest that the folding reaction of beta-LG follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the native state of beta-LG at pH 2 with more positive charges repulsion than at pH 7.5. Then TNB-beta-LG will become a useful model to analyze the conformation and stability of the intermediate of protein folding.     

Solid-state (13)C NMR chemical shift anisotropy tensors of polypeptides.

Carbon-13 chemical shift anisotropy (CSA) tensors for various carbon sites of polypeptides, and for carbon sites in alpha-helical and beta-sheet conformations of poly-L-alanine, and polyglycine, are presented. The carbonyl (13)C CSA tensors were determined from one-dimensional CPMAS spectra obtained at a slow spinning speed, whereas the CSA tensors of C(alpha) and other carbons in side chains of peptides were determined using 2D PASS experiments on powder samples. The results suggest that the spans of (13)Carbonyl CSA tensors of alanine and glycine residues in various peptides are similar, even though the magnitude of individual components of the CSA tensor and the isotropic chemical shift are different. In addition, the delta(22) element is the only component of the (13)Carbonyl CSA tensor that significantly depends on the CO.HN hydrogen-bond length. Solid-state NMR experimental results also suggest that (13)Carbonyl and (13)C(alpha) CSA tensors are similar for alpha-helical and beta-sheet conformations of poly-L-alanine, which is in agreement with the reported quantum chemical calculation studies and previous solid-state NMR experimental studies on other systems. On the other hand, the (13)C(alpha) CSA tensor of the first alanine residue is entirely different from that of the second or later alanine residues of the peptide. While no clear trends in terms of the span and the anisotropic parameter were predicted for (13)C(beta) CSA tensors of alanine, they mainly depend on the conformation and dynamics of the side chain as well as on the packing interactions in the solid state of peptides.     

Macrocyclic beta-sheet peptides that mimic protein quaternary structure through intermolecular beta-sheet interactions

This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.     

Mechanisms of the interaction of alpha-helical transmembrane peptides with phospholipid bilayers

The synthetic peptide acetyl-K(2)-G-L(24)-K(2)-A-amide (P(24)) and its analogs have been successfully utilized as models of the hydrophobic transmembrane alpha-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic alpha-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P(24) adopts a very stable alpha-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of alpha-helical transmembrane peptides with phospholipid bilayers are summarized here.     

Conformational equilibria of terminally blocked single amino acids at the water-hexane interface. A molecular dynamics study

The conformational equilibria of the acetyl and methyl amide terminally blocked L-alanine, L-leucine and L-glutamine amino acids are examined in vacuum, in bulk water, and at the water-hexane interface, using multi-nanosecond molecular dynamics simulations. The two-dimensional probability distribution functions of finding the peptides at different dihedral angles of the backbone, phi and psi, are calculated, and free energy differences between different conformational states are determined. All three peptides are interfacially active, i.e. tend to accumulate at the interface even though they are not amphiphilic. Conformational states stable in both gas phase and water are also stable in the interfacial environment. Their populations, however, cannot be simply predicted from the knowledge of conformational equilibria in the bulk phases, indicating that the interface exerts a unique effect on the peptides. Conformational preferences in the interfacial environment arise from the interplay between electrostatic and hydrophobic effects. As in an aqueous solution, electrostatic solute-solvent interactions lead to the stabilization of more polar peptide conformations. The hydrophobic effect is manifested at the interface by a tendency to segregate polar and nonpolar moieties of the solute into the aqueous and the hexane phases, respectively. For the terminally blocked glutamine, this favors conformations for which such a segregation is compatible with the formation of strong, backbone-side chain intramolecular hydrogen bonds on the hexane side of the interface. The influence of the hydrophobic effect can be also noted in the orientational preferences of the peptides at the interface. The terminally blocked leucine is oriented such that its nonpolar side chain is buried in hexane, whereas the polar side chain of glutamine is immersed in water. The free energies of rotating the peptides along the axis parallel to the interface by more than 90 degrees are substantial. This indicates that peptide folding at interfaces is strong by driven by the tendency to adopt amphiphilic structures.     

Solution structure, dimerization, and dynamics of a lipophilic alpha/3(10)-helical, C alpha-methylated peptide. Implications for folding of membrane proteins.

The solution structure and the dimerization behavior of the lipophilic, highly C(alpha)-methylated model peptide, mBrBz-Iva(1)-Val(2)-Iva(3)-(alphaMe)Val(4)-(alphaMe)Phe(5)-(alphaMe)Val(6)-Iva(7)-NHMe, was studied by NMR spectroscopy and molecular dynamics simulations. The conformational analysis resulted in a right-handed 3(10)/alpha-helical equilibrium fast on the NMR time scale with a slight preference for the alpha-helical conformation. The NOESY spectrum showed intermolecular NOEs due to an aggregation of the heptapeptide. In addition, temperature-dependent diffusion measurements were performed to calculate the hydrodynamic radius. All these findings are consistent with an antiparallel side-by-side dimerization. The structure of the dimeric peptide was calculated with a simulated annealing strategy. The lipophilic dimer is held together by favorable van der Waals interactions in the sense of a bulge fitting into a groove. The flexibility of the helical conformations concerning an alpha/3(10)-helical equilibrium is shown in a 3 ns molecular dynamics simulation of the resulting dimeric structure. Both overall helical structures of each monomer and the antiparallel mode of dimerization are stable. However, transitions were seen of several residues from a 3(10)-helical into an alpha-helical conformation and vice versa. Hence, this peptide represents a good model in which two often-discussed aspects of hierarchical transmembrane protein folding are present: i <-- i + 3 and i <-- i + 4 local H-bonding interactions cause a specific molecular shape which is then recognized as attractive by other surrounding structures.     

Entropic stabilization of isolated beta-sheets

Temperature-dependent electric deflection measurements have been performed for a series of unsolvated alanine-based peptides (Ac-WA(n)-NH(2), where Ac = acetyl, W = tryptophan, A = alanine, and n = 3, 5, 10, 13, and 15). The measurements are interpreted using Monte Carlo simulations performed with a parallel tempering algorithm. Despite alanine's high helix propensity in solution, the results suggest that unsolvated Ac-WA(n)-NH(2) peptides with n > 10 adopt beta-sheet conformations at room temperature. Previous studies have shown that protonated alanine-based peptides adopt helical or globular conformations in the gas phase, depending on the location of the charge. Thus, the charge more than anything else controls the structure.     

Elongation of ordered peptide aggregate of an amyloidogenic hexapeptide NFGAIL observed in molecular dynamics simulations with explicit solvent

The mechanisms by which amyloidogenic peptides and proteins form soluble toxic oligomers remain elusive. We have studied the formation of partially ordered tetramers and well-ordered octamers of an amyloidogenic hexapeptide NFGAIL (residues 22-27 of the human islet amyloid polypeptide) in our previous work. Continuing the effort, we here probe the beta-sheet elongation process by a combined total of 2.0 micros molecular dynamics simulations with explicit solvent. In a set of 10 simulations with the peptides restrained to the extended conformation, we observed that the main growth mode was elongation along the beta-sheet hydrogen bonds through primarily a two-stage process. Driven by hydrophobic forces, the peptides initially attached to the surface of the ordered oligomer, moved quickly to the beta-sheet edges, and formed stable beta-sheet hydrogen bonds. Addition of peptides to the existing oligomer notably improved the order of the peptide aggregate in which labile outer layer beta-sheets were stabilized, which provides good templates for further elongation. These simulations suggested that elongation along the beta-sheet hydrogen bonds occurs at the intermediate stage when low-weight oligomers start to form. We did not observe significant preference toward either parallel or antiparallel beta-sheets at the elongation stage for this peptide. In another set of 10 unrestrained simulations, the dominant growth mode was disordered aggregation. Taken together, these results offered a glimpse at the molecular events leading to the formation of ordered and disordered low-weight oligomers.     

Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of beta-lactoglobulin was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at special condition. The effect of n-alkyl sulfates on the structure of native beta-lactoglobulin at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of non-native alpha-helical intermediate. The addition of various concentrations of n-alkyl sulfates to the native state of beta-lactoglobulin (pH 2) appears to support the stabilized form of non-native alpha-helical intermediate at pH 2. The m values of the intermediate state of beta-lactoglobulin by SOS, SDeS, SDS and STS showed substantial variation. The enhancement of m values as the stability criterion of non-native alpha-helical intermediate state corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the non-native alpha-helical intermediate state.     

Multiple peptide conformations give rise to similar binding affinities: molecular simulations of p53-MDM2

Molecular dynamics simulations, guided by experimental information (Zondlo et al. Biochemistry 2006, 45, 11945-11957) have been used successfully to reproduce experimental trends in binding affinities of variant p53 peptides with MDM2. Simulations reveal how the conformations of the peptides and the receptor modulate each other to optimize interactions. The conformations of the uncomplexed peptides are governed by a combination of helix and intrinsic disorder (in agreement with experiments), while in the complexed state two very different conformations can coexist. This yields very similar binding affinities, driven by either enthalpy or entropy.     

Understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity

Small amphiphilic peptides are attractive building blocks to design biocompatible supramolecular structures via self-assembly, with applications in, for example, drug delivery, tissue engineering, and nanotemplating. We address the influence of systematical changes in the amino acid sequence of such peptides on the self-assembled macromolecular structures. For cationic-head surfactant-like eight-residue peptides, the apolar tail amino acids were chosen to systematically vary the propensity to form an alpha-helical secondary structure while conserving the overall hydrophobicity of the sequence. Characterization of the supramolecular structures indicates that for short peptides a beta-sheet secondary structure correlates with ribbonlike assemblies while random-coil and alpha-helical secondary structures correlate with assembly of rods.     

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.     

Tuning secondary structure and self-assembly of amphiphilic peptides

Self-assembly is one of nature's mechanisms by which higher order structures are obtained. Two of the main driving forces for self-assembly, hydrophobic interactions and hydrogen bonding, are both present within amphiphilic peptides. Here, it is demonstrated how the intricately interconnected folding and assembly behavior of an N-terminally acylated peptide, with the sequence GANPNAAG, has been tuned by varying its hydrophobic tail and thermal history. The change in interplay between hydrophobic forces and peptide folding allowed the occurrence of different types of aggregation, from soluble peptides with a random coil conformation to aggregated peptides arranged in a beta-sheet assembly, which form helically twisted bilayer ribbons.     

Symmetry-based constant-time homonuclear dipolar recoupling in solid state NMR

Constant-time dipolar recoupling pulse sequences are advantageous in structural studies by solid state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS) because they yield experimental data that are relatively insensitive to radio-frequency pulse imperfections and nuclear spin relaxation processes. A new approach to the construction of constant-time homonuclear dipolar recoupling sequences is described, based on symmetry properties of the recoupled dipole-dipole interaction Hamiltonian under cyclic displacements in time with respect to the MAS sample rotation period. A specific symmetry-based pulse sequence called PITHIRDS-CT is introduced and demonstrated experimentally. (13)C NMR data for singly-(13)C-labeled amino acid powders and amyloid fibrils indicate the effectiveness of PITHIRDS-CT in measurements of intermolecular distances in solids. (15)N-detected and (13)C-detected measurements of intramolecular (15)N-(15)N distances in peptides with alpha-helical and beta-sheet structures indicate the utility of PITHIRDS-CT in studies of molecular conformations, especially measurements of backbone psi torsion angles in peptides containing uniformly (15)N- and (13)C-labeled amino acids.     

Peptide folding using multiscale coarse-grained models

The multiscale coarse-graining (MS-CG) method has been previously used to describe the equilibrium properties of peptides. The present study reveals that MS-CG models of alpha-helical polyalanine and the beta-hairpin V 5PGV 5 possess the capacity to efficiently refold in simulations initiated from unfolded configurations. The MS-CG peptides exhibit free energy landscapes that are funneled toward folded configurations and two-state folding behavior, consistent with the known characteristics of small, rapidly folding peptides. Moreover, the models demonstrate enhanced sampling capabilities when compared to systems with full atomic detail. The significance of these observations with respect to the theoretical basis of the MS-CG approach is discussed. The MS-CG peptides were used to reconstruct atomically detailed configurations in order to evaluate the extent to which MS-CG ensembles embody all-atom peptide free energy landscapes. Ensembles obtained from these reconstructed configurations display good agreement with the all-atom simulation data used to generate the MS-CG models and also corroborate the presence of features observed in the MS-CG peptide free energy landscapes. These findings suggest that MS-CG models may be of significant utility in the study of peptide folding.     

Peptide recognition: encapsulation and alpha-helical folding of a nine-residue peptide within a hydrophobic dimeric capsule of a bowl-shaped host

A dimeric capsule of coordination bowl 1 encapsulated a nine-residue peptide (Trp-Ala-Glu-Ala-Ala-Ala-Glu-Ala-Trp; 2) within the large hydrophobic cavity in water, and stabilized the alpha-helical conformation of bound 2. An NMR titration experiment revealed that monomeric bowl 1 recognized two Trp residues at the both terminals of 2 through 1/2 = 1:1 to 2:1 complexation. The 1:1 and 2:1 species exist in equilibrium even in the presence of excess 1. It was found that the formation of the 2:1 complex, in which two bowls of 1 wrapped the whole of 2, became dominant by the addition of NaNO3 due to the fact that the enhanced ion strength increased the hydrophobic interaction between Trp residues and the cavity of 1. The alpha-helical conformation of 2 within the dimeric capsule of 1 was elucidated from detailed NOESY analysis.