Dihedral angles of tripeptides in solution directly determined by polarized Raman and FTIR spectroscopy

The amide I mode of the peptide linkage is highly delocalized in peptides and protein segments due to through-bond and through-space vibrationally coupling between adjacent peptide groups. J. Phys. Chem. B. 104:11316-11320) used coherent femtosecond infrared (IR) spectroscopy to determine the excitonic coupling energy and the orientational angle between the transition dipole moments of the interacting amide I modes of cationic tri-alanine in D(2)O. Recently, the same parameters were determined for all protonation states of tri-alanine by analyzing the amide I bands in the respective IR and isotropic Raman spectra (. J. Am. Chem. Soc. 119:1720-1726.). In both studies, the dihedral angles phi and psi were then obtained by utilizing the orientational dependence of the coupling energy obtained from ab initio calculations on tri-glycine in vacuo (. J. Raman Spectrosc. 29:81-86) to obtain an extended 3(1) helix-like structure for the tripeptide. In the present paper, a novel algorithm for the analysis of excitonic coupling between amide I modes is presented, which is based on the approach by Schweitzer-Stenner et al. but avoids the problematic use of results from ab initio calculations. Instead, the dihedral angles are directly determined from infrared and visible polarized Raman spectra. First, the interaction energy and the corresponding degree of wave-function mixing were obtained from the amide I profile in the isotropic Raman spectrum. Second, the depolarization ratios and the amide I profiles in the anisotropic Raman and IR-absorption spectra were used to determine the orientational angle between the peptide planes and the transition dipole moments, respectively. Finally, these two geometric parameters were utilized to determine the dihedral angles phi and psi between the interacting peptide groups. Stable extended conformations with dihedral angles in the beta-sheet region were obtained for all protonation states of tri-alanine, namely phi(+) = -126 degrees, psi(+) = 178 degrees; phi(+/-) = -110 degrees, psi(+/-) = 155 degrees; and phi(-) = -127 degrees, psi(-) = 165 degrees for the cationic, zwitterionic, and anionic state, respectively. These values reflect an extended beta-helix structure. Tri-glycine was found to be much more heterogeneous in that different extended conformers coexist in the cationic and zwitterionic state, which yield a noncoincidence between isotropic and anisotropic Raman scattering. Our study introduces vibrational spectroscopy as a suitable tool for the structure analysis of peptides in solution and tripeptides as suitable model systems for investigating the role of local interactions in determining the propensity of peptide segments for distinct secondary structure motifs.     

Flexibility of bovine pancreatic trypsin inhibitor

The native conformation of a protein may be expressed in terms of the dihedral angles, phi's and psi's for the backbone, and kappa's for the side chains, for a given geometry (bond lengths and bond angles). We have developed a method to obtain the dihedral angles for a low-energy structure of a protein, starting with the X-ray structure; it is applied here to examine the degree of flexibility of bovine pancreatic trypsin inhibitor. Minimization of the total energy of the inhibitor (including nonbonded, electrostatic, torsional, hydrogen bonding, and disulfide loop energies) yields a conformation having a total energy of -221 kcal/mol and a root mean square deviation between all atoms of the computed and experimental structures of 0.63 A. The optimal conformation is not unique, however, there being at least two other conformations of low-energy (-222 and -220 kcal/mol), which resemble the experimental one (root mean square deviations of 0.66 and 0.64 A, respectively). These three conformations are located in different positions in phi, psi space, i.e., with a total deviation of 81 degrees, 100 degrees and 55 degrees from each other (with a root mean square deviation of several degrees per dihedral angle from each other). The nonbonded energies of the backbones, calculated along lines in phi, psi space connecting these three conformations, are all negative, without any intervening energy barriers (on an energy contour map in the phi, psi plane). Side chains were attached at several representative positions in this plane, and the total energy was minimized by varying the kappa's. The energies were of approximately the same magnitude as the previous ones, indicating that the conformation of low energy is flexible to some extent in a restricted region of phi, psi space. Interestingly, the difference delta phi i+1 in phi i+1 for the (i + 1)th residue from one conformation to another is approximately the same as -delta psi i for the ith residue; i.e., the plane of the peptide group between the ith and (i + 1)th residues re-orient without significant changes in the positions of the other atoms. The flexibility of the orientations of the planes of the peptide groups is probably coupled in a cooperative manner to the flexibility of the positions of the backbone and side-chain atoms.     

Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR

The orientation of a beta-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) (15)N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic beta-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating (15)N-(1)H dipolar coupling with (15)N chemical shift to determine the orientation of multiply (15)N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the beta-strand axis and the rotation of the beta-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20 degrees +/- 10 degrees from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65 degrees +/- 15 degrees . These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (phi, psi) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal beta-hairpin with uniform (phi, psi) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the beta-sheet curvature but does not make it ideal. (31)P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this beta-hairpin antimicrobial peptide.     

Strategy for supplementing structure calculations using limited data with hydrophobic distance restraints

Introductory biochemistry texts often note that the fold of a protein is completely defined when the dihedral angles phi and psi are known for each amino acid. This assertion was examined with torsion angle dynamics and simulated annealing (TAD/SA) calculations of protein G using only dihedral angle restraints. When all dihedral angles were restrained to within 1 degrees of the values of the X-ray structure, the TAD/SA structures gave a backbone root mean square deviation to the target of 4 A. Factors that contributed to divergence from the correct solution include deviations of peptide bonds from planarity, internal conflicts resulting from the nonuniform energies of different phi, psi combinations, and relaxation to extended conformations in the absence of long-range constraints. Simulations including hydrogen-bond restraints showed that even a few long-range contacts constrain the fold better than a complete set of accurate dihedral restraints. A procedure is described for TAD/SA calculations using hydrogen-bond restraints, idealized dihedral restraints for residues in regular secondary structures, and "hydrophobic distance restraints" derived from the positions of hydrophobic residues in the amino acid sequence. The hydrogen-bond restraints are treated as inviolable, whereas violated hydrophobic restraints are removed following reduction of restraint upper bounds from 2 to 1 times the predicted radius of gyration. The strategy was tested with simulated restraints from X-ray structures of proteins from different fold classes and NMR data for cold shock protein A that included only backbone chemical shifts and hydrogen bonds obtained from a long-range HNCO experiment.     

FTIR reveals structural differences between native beta-sheet proteins and amyloid fibrils

The presence of beta-sheets in the core of amyloid fibrils raised questions as to whether or not beta-sheet-containing proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the molecular structure of amyloid fibrils differs more generally from the beta-sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the phi/psi dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the beta-sheet twist. These data imply that amyloid fibril formation from native beta-sheet proteins can involve a substantial structural reorganization.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

3(10)-helices in proteins are parahelices

The 3(10)-helix is characterized by having at least two consecutive hydrogen bonds between the main-chain carbonyl oxygen of residue i and the main-chain amide hydrogen of residue i + 3. The helical parameters--pitch, residues per turn, radius, and root mean square deviation (rmsd) from the best-fit helix--were determined by using the HELFIT program. All 3(10)-helices were classified as regular or irregular based on rmsd/(N - 1)1/2 where N is the helix length. For both there are systematic, position-specific shifts in the backbone dihedral angles. The average phi, psi shift systematically from approximately -58 degrees, approximately -32 degrees to approximately -90 degrees, approximately -4 degrees for helices 5, 6, and 7 residues long. The same general pattern is seen for helices, N = 8 and 9; however, in N = 9, the trend is repeated with residues 6, 7, and 8 approximately repeating the phi, psi of residues 2, 3, and 4. The residues per turn and radius of regular 3(10)-helices decrease with increasing length of helix, while the helix pitch and rise per residue increase. That is, regular 3(10)-helices become thinner and longer as N increases from 5 to 8. The fraction of regular 3(10)-helices decreases linearly with helix length. All longer helices, N > or = 9 are irregular. Energy minimizations show that regular helices become less stable with increasing helix length. These findings indicate that the definition of 3(10)-helices in terms of average, uniform dihedral angles is not appropriate and that it is inherently unstable for a polypeptide to form an extended, regular 3(10)-helix. The 3(10)-helices observed in proteins are better referred to parahelices.     

Predominant torsional forms adopted by dipeptide conformers in solution: parameters for molecular recognition.

The present paper describes the predominant conformational forms adopted by dipeptides in aqueous solution. More than 50 dipeptides were subjected to conformational analysis using SYBYL Random Search. The resultant collections of conformers for individual dipeptides, for small groups with related side chain residues and for large groups of about 50 dipeptides were visualized graphically and analysed using a novel three-dimensional pseudo-Ramachandran plot. The distribution of conformers, weighted according to the percentage of each in the total conformer pool, was found to be restricted to nine main combinations of backbone psi (psi) and phi (phi) torsion angles. The preferred psi values were in sectors A7 (+150 degrees to +/-180 degrees), A10 (+60 degrees to +90 degrees) and A4 (-60 degrees to -90 degrees), and these were combined with preferred phi values in sectors B12 (-150 degrees to +/-180 degrees), B9 (-60 degrees to -90 degrees) and B2 (+30 degrees to +60 degrees). These combinations of psi and phi values are distinct from those found in common secondary structures of proteins. These results show that although dipeptides can each adopt many conformations in solution, each possesses a profile of common conformers that is quantifiable. A similarly weighted distribution of dipeptide conformers according to distance between amino-terminal nitrogen and carboxyl-terminal carbon shows how the preferred combinations of backbone torsional angles result in particular N-C geometries for the conformers. This approach gives insight into the important conformational parameters of dipeptides that provide the basis for their molecular recognition as substrates by widely distributed peptide transporters. It offers a basis for the rational design of peptide-based bioactive compounds able to exploit these transporters for targeting and delivery.     

NMR structures of a nonapeptide from DNA binding domain of human polymerase-alpha determined by iterative complete-relaxation-matrix approach.

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-alpha, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by alpha, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (-3.1 x 10(-3)) of the fourth residue, 3J(NHalpha) coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, phi and psi dihedral angles for the i + 1, and i + 2 residues of the turn are found to be -80 and -41 and -60 and -40 degrees. The first proline residue is trans- while the second exists in both cis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5alpha-P6alpha correlation and phi and psi angles of the proline. The five-membered proline ring is in DOWN puckered (C-beta-exo/C-gamma-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are approximately 5 A(o) apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.     

Structures, basins, and energies: a deconstruction of the Protein Coil Library

Globular proteins adopt complex folds, composed of organized assemblies of alpha-helix and beta-sheet together with irregular regions that interconnect these scaffold elements. Here, we seek to parse the irregular regions into their structural constituents and to rationalize their formative energetics. Toward this end, we dissected the Protein Coil Library, a structural database of protein segments that are neither alpha-helix nor beta-strand, extracted from high-resolution protein structures. The backbone dihedral angles of residues from coil library segments are distributed indiscriminately across the phi,psi map, but when contoured, seven distinct basins emerge clearly. The structures and energetics associated with the two least-studied basins are the primary focus of this article. Specifically, the structural motifs associated with these basins were characterized in detail and then assessed in simple simulations designed to capture their energetic determinants. It is found that conformational constraints imposed by excluded volume and hydrogen bonding are sufficient to reproduce the observed ,psi distributions of these motifs; no additional energy terms are required. These three motifs in conjunction with alpha-helices, strands of beta-sheet, canonical beta-turns, and polyproline II conformers comprise approximately 90% of all protein structure.     

The normal modes of the gramicidin-A dimer channel.

The dynamics of the gramicidin-A dimer channel is studied in the harmonic approximation by a vibrational analysis of the atomic motions relative to their equilibrium positions. The system is represented by an empirical potential energy function, and all degrees of freedom (bonds lengths, bond angles, and torsional angles) are allowed to vary. The thermal fluctuations in the backbone dihedral angles phi and psi, atomic root mean square displacements, and the correlations between the different amide planes are computed. It is found that only adjacent dihedral psi i and phi i+1 are strongly correlated, while different hydrogen-bonded amide planes are only weakly correlated. Modes with relatively low vibrational frequencies (75-175 cm-1) make the dominant contributions to the carbonyl librations. The general flexibility of the structure and the role of carbonyl librations in the ion transport mechanism are discussed.     

Characterization of beta-turn and Asx-turns mimicry in a model peptide: stabilization via C--H . . . O interaction

The chemical synthesis and single-crystal X-ray diffraction analysis of a model peptide, Boc-Thr-Thr-NH2 (1) comprised of proteinogenic residues bearing an amphiphilic Cbeta -stereogenic center, has been described. Interestingly, the analysis of its molecular structure revealed the existence of a distinct conformation that mimics a typical beta-turn and Asx-turns, i.e., the two Thr residues occupy the left- and right-corner positions. The main-chain torsion angles of the N- and C-terminal residues i.e., semiextended: phi = -68.9 degrees , psi = 128.6 degrees ; semifolded: phi = -138.1 degrees , psi = 2.5 degrees conformations, respectively, in conjunction with a gauche- disposition of the obligatory C-terminus Thr CgammaH3 group, characterize the occurrence of the newly described beta-turn- and Asx-turns-like topology. The preferred molecular structure is suggested to be stabilized by an effective nonconventional main-chain to side-chain Ci=O . . . H--Cgamma(i+2)-type intraturn hydrogen bond. Noteworthy, the observed topology of the resulting 10-membered hydrogen-bonded ring is essentially similar to the one perceived for a classical beta-turn and the Asx-turns, stabilized by a conventional intraturn hydrogen bond. Considering the signs as well as magnitudes of the backbone torsion angles and the orientation of the central peptide bond, the overall mimicked topology resembles the type II beta-turn or type II Asx-turns. An analysis of Xaa-Thr sequences in high-resolution X-ray elucidated protein structures revealed the novel topology prevalence in functional proteins (unpublished). In view of indubitable structural as well as functional importance of nonconventional interactions in bioorganic and biomacromolecules, we intend to highlight the participation of Thr CgammaH in the creation of a short-range C=O . . . H--Cgamma -type interaction in peptides and proteins.     

Ab initio computational study of beta-cellobiose conformers using B3LYP/6-311++G**

The molecular structure of 27 conformers of beta-cellobiose were studied in vacuo through gradient geometry optimization using B3LYP density functionals and the 6-311++G** basis set. The conformationally dependent geometry changes and energies were explored as well as the hydrogen-bonding network. The lowest electronic energy structures found were not those suggested from available crystallographic and NMR solution data, where the glycosidic dihedral angles fall in the region (phi, psi) approximately (40 degrees, -20 degrees ). Rather, 'flipped' conformations in which the dihedral angles are in the range (phi, psi) approximately (180 degrees, 0 degrees ) are energetically more stable by approximately 2.5 kcal/mol over the 'experimentally accepted' structure. Further, when the vibrational free energy, deltaG, obtained from the calculated frequencies, is compared throughout the series, structures with (phi, psi) in the experimentally observed range still have higher free energy ( approximately 2.0 kcal/mol) than 'flipped' forms. The range of bridging dihedral angles of the 'normal' conformers, resulting from the variance in the phi dihedral is larger than that found in the 'flipped' forms. Due to this large flat energy surface for the normal conformations, we surmise that the summation of populations of these conformations will favor the 'normal' conformations, although evidence suggests that polar solvent effects may play the dominant role in providing stability for the 'normal' forms. Even though some empirical studies previously found the 'flipped' conformations to be lowest in energy, these studies have been generally discredited because they were in disagreement with experimental results. Most of the DFT/ab initio conformations reported here have not been reported previously in the ab initio literature, in part because the use of less rigorous theoretical methods, i.e. smaller basis sets, have given results in general agreement with experimental data, that is, they energetically favored the 'normal' forms. These are the first DFT/ab initio calculations at this level of theory, apparently because of the length and difficulty of carrying out optimizations at these high levels.     

Structure and conformation of linear peptides. I. Structure of L-tyrosyl-L-tyrosine

L-tyrosyl-L-tyrosine crystallizes as a dihydrate in the orthorhombic system, space group C222(1), with a = 12.105(2), b = 12.789(2), c = 24.492(3) A, Z = 8. The structure was solved by direct methods and refined to a final R-value of 0.059 for 1740 observed reflections. The molecule exists as a zwitterion, the peptide unit is trans planar, and the backbone torsion angles correspond to an extended conformation, with psi 1 = 149.4 degrees, phi 2 = -161.2 degrees, psi 2 = 158.3 degrees. The values of the side-chain torsion angles (chi 1, chi 2) are (-58.8 degrees, -63.1 degrees) for the first tyrosine and (-171.7 degrees, -116.5 degrees) for the second. The planes of the aromatic rings are nearly parallel (dihedral angle of 6.1 degrees), and their centers are separated by 10.9 A. The carboxyl plane forms a dihedral angle of 23.8 degrees with the plane of the peptide bond.     

Crystal structure of tert.-butyloxycarbonyl-L-prolyl-D-alanyl-D-alanyl-N-methylamide. Dimeric beta-sheet formation.

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH3, monohydrate, (C17H30N4O5.H2O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are monoclinic, space group P2(1), a = 9.2585(4), b = 9.3541(5), c = 12.4529(4)A, beta = 96.449(3) degrees, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (delta omega = 5.2 degrees and delta omega = 3.7 degrees, respectively). The backbone torsion angles are: phi 1 = -60 degrees, psi 1 = 143.3 degrees, omega 1 = -174.8 degrees, phi 2 = 148.4 degrees, psi 2 = -143.1 degrees, omega 2 = -179.7 degrees, phi 3 = 151.4 degrees, psi 3 = -151.9 degrees, omega 3 = -176.3 degrees. The pyrrolidine ring of the proline residue adopts the C2-C gamma conformation. The molecular packing gives rise to an antiparallel beta-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric beta-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient beta-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH3.     

Polypeptide motions are dominated by peptide group oscillations resulting from dihedral angle correlations between nearest neighbors

To identify basic local backbone motions in unfolded chains, simulations are performed for a variety of peptide systems using three popular force fields and for implicit and explicit solvent models. A dominant "crankshaft-like" motion is found that involves only a localized oscillation of the plane of the peptide group. This motion results in a strong anticorrelated motion of the phi angle of the ith residue (phi(i)) and the psi angle of the residue i - 1 (psi(i-1)) on the 0.1 ps time scale. Only a slight correlation is found between the motions of the two backbone dihedral angles of the same residue. Aside from the special cases of glycine and proline, no correlations are found between backbone dihedral angles that are separated by more than one torsion angle. These short time, correlated motions are found both in equilibrium fluctuations and during the transit process between Ramachandran basins, e.g., from the beta to the alpha region. A residue's complete transit from one Ramachandran basin to another, however, occurs in a manner independent of its neighbors' conformational transitions. These properties appear to be intrinsic because they are robust across different force fields, solvent models, nonbonded interaction routines, and most amino acids.     

A survey of left-handed polyproline II helices

Left-handed polyproline II helices (PPII) are contiguous elements of protein secondary structure in which the phi and psi angles of constituent residues are restricted to around -75 degrees and 145 degrees, respectively. They are important in structural proteins, in unfolded states and as ligands for signaling proteins. Here, we present a survey of 274 nonhomologous polypeptide chains from proteins of known structure for regions that form these structures. Such regions are rare, but the majority of proteins contain at least one PPII helix. Most PPII helices are shorter than five residues, although the longest found contained 12 amino acids. Proline predominates in PPII, but Gln and positively charged residues are also favored. The basis of Gln's prevalence is its ability to form an i, i + 1 side-chain to main-chain hydrogen bond with the backbone carbonyl oxygen of the proceeding residue; this helps to fix the psi angle of the Gln and the phi and psi of the proceeding residue in PPII conformations and explains why Gln is favored at the first position in a PPII helix. PPII helices are highly solvent exposed, which explains why apolar amino acids are disfavored despite preferring this region of phi/psi space when in isolation. PPII helices have perfect threefold rotational symmetry and within these structures we find significant correlation between the hydrophobicity of residues at i and i + 3; thus, PPII helices in globular proteins can be considered to be amphipathic.     

Ab initio construction of polypeptide fragments: efficient generation of accurate,representative ensembles

We describe a novel method to generate ensembles of conformations of the main-chain atoms [N, C(alpha), C, O, Cbeta] for a sequence of amino acids within the context of a fixed protein framework. Each conformation satisfies fundamental stereo-chemical restraints such as idealized geometry, favorable phi/psi angles, and excluded volume. The ensembles include conformations both near and far from the native structure. Algorithms for effective conformational sampling and constant time overlap detection permit the generation of thousands of distinct conformations in minutes. Unlike previous approaches, our method samples dihedral angles from fine-grained phi/psi state sets, which we demonstrate is superior to exhaustive enumeration from coarse phi/psi sets. Applied to a large set of loop structures, our method samples consistently near-native conformations, averaging 0.4, 1.1, and 2.2 A main-chain root-mean-square deviations for four, eight, and twelve residue long loops, respectively. The ensembles make ideal decoy sets to assess the discriminatory power of a selection method. Using these decoy sets, we conclude that quality of anchor geometry cannot reliably identify near-native conformations, though the selection results are comparable to previous loop prediction methods. In a subsequent study (de Bakker et al.: Proteins 2003;51:21-40), we demonstrate that the AMBER forcefield with the Generalized Born solvation model identifies near-native conformations significantly better than previous methods.     

Membrane-bound conformation and topology of the antimicrobial peptide tachyplesin I by solid-state NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.     

Structure and conformation of linear peptides. VIII. Structure of t-Boc-glycyl-L-phenylalanine

The crystal structure of t-Boc-glycyl-L-phenylalanine (C14H22N2O5, molecular weight = 298) has been determined. Crystals are monoclinic, space group P2(1), with a = 7.599(1) A, b = 9.576(2), c = 12.841(2), beta = 97.21(1) degrees, Z = 2, Dm = 1.149, Dc = 1.168 g X cm-3. Trial structure was obtained by direct methods and refined to a final R-index of 0.064 for 1465 reflections with I greater than 1 sigma. The peptide unit is trans planar and is nearly perpendicular to the plane containing the urethane moiety. The plane of the carboxyl group makes a dihedral angle of 16.0 degrees with the peptide unit. The backbone torsion angles are omega 0 = -176.9 degrees, phi 1 = -88.0 degrees, psi 1 = -14.5 degrees, omega 1 = 176.4 degrees, phi 2 = -164.7 degrees and psi 2 = 170.3 degrees. The phenylalanine side chain conformation is represented by the torsion angles chi 1 = 52.0 degrees, chi 2 = 85.8 degrees.