Crystal structure and conformation of a cyclic tetrapeptide cyclo(L-Pro-Sar)2 containing all-cis peptide units

The crystal structure and conformation of the synthetic cyclic tetrapeptide, cyclo(L -Pro-Sar)₂, was determined by x-ray analysis. The peptide crystallizes in the orthorhombic space group P2₁2₁2₁ with cell parameters a = 9.277(1), b = 12.884(1), and c = 15.581(2) Å. The crystal structure was solved by the symbolic addition procedure for direct phase determination and least-squares refinement using 1796 reflections, which led to the final R value of 0.043. This structure provides the first example observed in a crystal of a cyclic tetrapeptide in which all four peptide units have been found in the cis conformation with ω angles deviating slightly by 2°–10° from the ideal value of 0°. It was also found that the two Pro C^α-CO single bonds assumed a trans′ (ψ = 159.6° and 158.4°) conformation. Adjoining average planes of the peptide groups fall at nearly right angles to each other. The pyrrolidine ring conformations of the two prolyl residues are in the envelope form, with C^γ carbon out of the least-squares planes for the remaining four atoms.     

Effect of urea on peptide conformation in water: molecular dynamics and experimental characterization

Molecular dynamics simulations of a ribonuclease A C-peptide analog and a sequence variant were performed in water at 277 and 300 K and in 8 M urea to clarify the molecular denaturation mechanism induced by urea and the early events in protein unfolding. Spectroscopic characterization of the peptides showed that the C-peptide analog had a high alpha-helical content, which was not the case for the variant. In the simulations, interdependent side-chain interactions were responsible for the high stability of the alpha-helical C-peptide analog in the different solvents. The other peptide displayed alpha-helical unwinding that propagated cooperatively toward the N-terminal. The conformations sampled by the peptides depended on their sequence and on the solvent. The ability of water molecules to form hydrogen bonds to the peptide as well as the hydrogen bond lifetimes increased in the presence of urea, whereas water mobility was reduced near the peptide. Urea accumulated in excess around the peptide, to which it formed long-lived hydrogen bonds. The unfolding mechanisms induced by thermal denaturation and by urea are of a different nature, with urea-aqueous solutions providing a better peptide solvation than pure water. Our results suggest that the effect of urea on the chemical denaturation process involves both the direct and indirect mechanisms.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

Backbone conformational dependence of peptide acidity

Electrostatic interactions at the protein surface yield over a billion-fold range of amide hydrogen exchange rates. This range is equivalent to the maximal degree of attenuation in exchange rates that have been shown to occur for amides buried within the protein interior. Continuum dielectric analysis of Ala-Ala, Ala-Gly, Gly-Ala and trans-Pro-Ala peptide conformer acidities predicts that the relative orientation of the two neighboring peptide groups can account for a million-fold variation in hydroxide-catalyzed hydrogen exchange rates. As in previous protein studies, an internal dielectric value of 3 was found to be applicable to simple model peptides, presumably reflecting the short lifetime of the peptide anion intermediate. Despite the million-fold range in conformer acidities, the small differences in the experimental exchange rates for these peptides are accurately predicted. Ala-Ala conformers with an extended N-terminal residue and the C-terminal residue in the α conformation are predicted to account for over 60% of the overall hydrogen exchange reaction, despite constituting only 12% of the protein coil population.     

Parallel tempering molecular dynamics folding simulation of a signal peptide in explicit water

Parallel temperature molecular dynamics simulations are used to explore the folding of a signal peptide, a short but functionally independent domain at the N-terminus of proteins. The peptide has been analyzed previously by NMR, and thus a solid reference state is provided with the experimental structure. Particular attention is paid to the role of water considered in full atomic detail. Different partial aspects in the folding process are quantified. The major group of obtained structures matches the NMR structure very closely. An important biological consequence is that in vivo folding of signal peptides seems to be possible within aqueous environments.     

Studies on the structure and conformation of yeast mitochondrial ATPase using aurovertin and methanol as probes

1. The isolation of the mitochondrial ATPase F1 and its beta-subunit from commercial baker's yeast (Saccharomyces cerevisiae) is described. 2. The molecular weight determined by ultracentrifugation is 340000 +/- 30000. Gel chromatography indicates a molecular weight of 300000 +/- 20000. 3. Fluorimetric titration of the isolated enzyme with aurovertin reveals two binding sites per molecule. The isolated beta-subunit binds aurovertin in a 1 : 1 stoicheiometry. It is concluded that the ATPase molecule contains two aurovertin-binding beta-subunits. 4. The stabilizing agent methanol influences both the measured Kd and the concentration of binding sites for aurovertin. These results fit a model in which both F1 and aurovertin are distributed between aqueous and methanol phases. 5. The effect of methanol on the ATPase activity can be described in terms of the model proposed by Recktenwald and Hess (Recktenwald, D. and Hess, B. (1977) FEBS Lett. 76, 25-28). It is proposed that methanol enhances the affinity of the regulatory site for ATP, but at higher concentrations prevents the interaction between the regulatory and catalytic sites. 6. Since HSO(-3), a typical effector of the assumed regulatory site of F1, has no effect on the binding of aurovertin, it is concluded that the binding site of aurovertin is not correlated with the regulatory site. 7. The inhibition of ATPase activity by aurovertin is slowly (t 1/2 = 70 s) induced during turnover conditions. 8. From the effect of methanol on the inhibition of ATPase activity by aurovertin it is concluded that under turnover conditions the conformation is such that the aurovertin-binding sites have a 6-fold higher affinity for methanol than under resting conditions.     

Folded conformation of an immunostimulating tetrapeptide rigin: high temperature molecular dynamics simulation study

Employing high temperature quenched molecular dynamics (QMD) simulations the conformational energy space of an immunostimulating tetrapeptide rigin: H-Gly³⁴¹-Gln-Pro-Arg³⁴⁴-OH, is explored. Using distance dependent dielectric (=r_ij) 31 different low energy starting structures with identical sequence were computed for their conformational preferences. According to the hypothesis of O'Connors et al. [J. Med. Chem. 35 (1992), 2870], 83 low-energy conformers resulted from unrestrained molecular dynamics (MD) simulations, could be classified into two energy minimized families: A and B, comprised of 64 (Pro C^γ-endo orientation) and 19 (Pro C^γ-exo orientation) structures, respectively. An examination of these families revealed the existence of a remarkably similar folded backbone conformation: torsion angles being φ_i+1 ≈−65°, ψ_i+1 ≈−65°, φ_i+2 ≈−65°, ψ_i+2 ≈−60°, characterizing a distorted type III β-turn structure across the central Gln-Pro segment. The folded conformation of rigin is devoid of a classical 1 ← 4 intra-molecular hydrogen bond nevertheless, the conformation is stabilized by an effective ‘salt-bridge’, i.e., Gly H₃N⁺… COO⁻ Arg interaction. Surprisingly, in both the families the unusual folded side-chain dispositions of the Gln residue favor the formation of a unique intra-residue ‘main-chain to side-chain’ H-bond, i.e., N–H…N^ε interaction, encompassing a seven-membered ring motif. The conformational attributes may be valuable in de novo construction of structure-based drug candidates having sufficient stimulating activity.     

Studies on proline-containing tetrapeptide models of beta-turns

The synthesis of a series of protected tetrapeptides of the general formula Cbz-Gly-X²-Y³-Gly-OR (R = stearyl or methyl, X and/or Y = proline) is described. Detailed CD studies have been performed to evaluate the contribution of proline-containing β-turns to the CD spectra of proteins. The CD spectra of all the models are dominated by the chiral contribution of the proline residue. In polar, proton-donating solvents, a poly-proline II-like spectrum was observed in almost all cases. The tetrapeptide model Cbz-Gly-Gly-Pro-Gly-OStearyl, in acetonitrile shows a type C spectrum that has not been previously reported for linear peptides. The ir and nmr data on this model support the assumption of that of a type III β-turn, exhibiting a type C spectrum, participate in the conformational equilibrium. The most interesting finding of the CD studies is the observation of a type D spectrum (according to the classification of Woody [Woody, R. W. (1974) in Peptides, Polypeptides and Proteins, Blout, E. R. Bovey, F. A. Lotan, N. & Goodman, M. (Eds.), Wiley, New York]) for models Cbz-Gly-Pro-Asp(OBu^t)-Gly-OStearyl and Cbz-Gly-Pro-Ser(OBu^t)-Gly-OStearyl in cyclohexane. The results of the CD measurements ae discussed in correlation with ir and nmr data and with recent literature.     

The preferred conformation of the tripeptide Ala-Phe-Ala in water is an inverse gamma-turn: implications for protein folding and drug design

Recent studies have provided evidence that peptides as short as tripeptides do adopt preferred conformations. Here we report that the tripeptide Ala-Phe-Ala (AFA) in aqueous solution preferentially forms an inverse gamma-turn. Circular dichroism (CD) indicated the presence of a predominant turn structure, and Fourier transform infrared (FTIR) bands suggested the presence of a gamma-turn forming a bifurcated H-bond with the solvent molecules. The high-resolution structure was obtained by a combined use of NMR spectroscopy and calculations. On the basis of 30 unambiguous ROESY-derived distance restraints (including the Halpha-NH NOE between Ala(1) and Ala(3) and a hydrogen bond between the CO group of Ala(1) and the NH group of Ala(3)), calculations clearly demonstrated the presence of an inverse gamma-turn centered on Phe(2). From NOE data, we estimated a mole fraction for the gamma-turn of 0.65. Since for AFA an extended beta-strand was also reported [Eker, F., Griebenow, K., Cao, X., Nafie, L. A., and Schweitzer-Stenner, R. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 10054-10059], we investigated the possibility that gamma-turn and beta-strand may represent two major conformations. By using a best-fit procedure that calculated experimental NOEs as weighted averages of the effects originating from both structures, we were able to calculate with good accuracy the backbone NOEs at 280 K in terms of the two limiting conformers, yielding a mole fraction for the gamma-turn and beta-strand conformations of 0.60 and 0.40, respectively, in good agreement with those found by NOE data. The implication of the existence of a preferred conformation by a small structural element is discussed in the context of the nucleation of protein folding events and the design of small peptide and peptidomimetic drugs.     

The folding and solution conformation of penicillin G acylase

The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by near- and far-ultraviolet circular dichroism, steady-state and time-resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid-point of 4.5 M urea. Separation of its constituent alpha and beta peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated beta peptide aggregates over a wide range of urea concentrations but that the alpha peptide refolds reversibly to a compact state. Physical studies showed that the refolded alpha peptide has a compact but asymmetric structure with more alpha helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8-anilino-1-naphthalene sulfonic acid binding and which is normally covered by the beta peptide in the native enzyme. The results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.     

Stereochemical control of peptide folding

Stereochemically constrained amino acid residues that strongly favour specific backbone conformations may be used to nucleate and stabilize specific secondary structures in designed peptides. An overview of the use of alphaalpha-dialkyl amino acids in stabilizing helical structures in synthetic peptides is presented, with an emphasis on work carried out in the authors laboratory. Alpha-aminoisobutyric acid (Aib) and related achiral homologs facilitate stable helix formation in oligopeptides as exemplified by a large number of crystal structure determinations in the solid state. The ability to design conformationally rigid helical modules has been exploited in attempts to design structurally well characterized helix-linker helix, using potential nonhelical linking segments. Beta-hairpin design has been approached by exploiting the tendency of 'prime turns' to nucleate hairpin formation. The use of nucleating (D)Pro-Gly segments has resulted in the generation of several well characterized beta-hairpin structures, including the crystallographic observation of beta-hairpin in a synthetic apolar octapeptide. Extensions of this approach to three stranded beta-sheets and larger structures containing multiple (D)Pro-Gly segments appear readily possible.     

Proline peptide isomerization and protein folding

The unfolding-refolding of proteins is a cooperative process and, as judged by equilibrium properties, occurs in one step involving the native,N, and the unfoldedU, conformational states. Kinetic studies have shown that the denatured protein exists as a mixture of slow-(U)_Sand fast-(U)_Frefolding forms produced by proline peptidecis-trans isomerization. Proline residues inU _Fare in the same configuration as in the native protein while they are in non-native configuration inU _S. For protein folding to occur quicklyU _Smust be converted intoU _F. The fact that the equilibrium and kinetic properties of are the same as those found for prolinecis-trans isomerization taken together with the absence of slow phase in the kinetics of refolding of a protein devoid of proline, support this view. However, the absence of a linear correlation between half-time of reactivation of denatured enzymes and their proline-contents, as well as the dissimilarities in the kinetic properties of in unfolding and refolding experiments are not consistent with the model. Conformational energy calculation and experimental results on refolding of proteins suggest that some proline residues are non-essential. They will not block protein folding even in wrong isomeric form. The native-like folded structure with incorrect proline isomers will serve as intermediate state(s) in which these prolines will more readily isomerize to the correct isomeric form. The picture becomes more complex when one considers the consequence ofcis-trans isomerism of non-proline residues on protein folding.     

Methods of peptide conformation studies

In solution most of the peptides assume multiple flexible conformations. Determination of the dominant conformers and evaluation of their populations is the aim of peptide conformation studies, in which theoretical and experimental methods play complementary roles. Molecular dynamics or Monte Carlo methods are quite effective in searching the conformational space accessible to a peptide but they are not able to estimate, precisely enough, the populations of various conformations. Therefore, they must be supplemented by experimental data. In this paper, a short review of the experimental methods, most widely used in peptide conformational studies, is presented. Among them NMR plays the leading role. Valuable information is also obtained from hydrogen exchange, fluorescence resonance energy transfer, and circular dichroism measurements. The advantages and shortcomings of these methods are discussed.     

N-acyl-L-phenylalanine derivatives as potent VLA-4 antagonists that mimic a cyclic peptide conformation.

A series of N-benzylpyroglutamyl-L-phenylalanine derivatives bearing carbamoyl substituents in the 3- or 4-positions was prepared and assayed for inhibition of the interaction between VCAM and VLA-4. Potent inhibition was observed in a number of analogues with substitution in the 4-position favored over the 3-position. A crystal structure of the key intermediate 25 indicates that it accesses a low energy conformation which closely matches key pharmacophores of a structurally characterized cyclic peptide.     

Reductive N,N-dimethylation of amino acid and peptide derivatives using methanol as the carbonyl source

Formaldehyde is readily formed from methanol in the presence of palladium-on-charcoal and air. The use of this methanolic formaldehyde solution for the reductive methylation of amino groups in amino acid and peptide derivatives by catalytic hydrogenation has been studied and found to be superior to the use of aqueous formaldehyde because no contaminating paraformaldehyde is present. Some data on N-isopropylation are given.