Conformational studies on peptides with proline in the right-handed alpha-helical region

The proline residues in proteins are known to play an important structural role. Recently, the role of a proline residue in the middle of right-handed alpha-helical segments of peptides has been the focus of attention. This role seems to be particularly important in the case of membrane proteins and in the tight packing of globular proteins. In the present study the right-handed alpha-helical region of the Ala-Pro dipeptide and of polypeptides containing this group have been investigated. Crystal structures of proline-containing alpha-helices from some proteins have been analyzed and energy minimization studies on some model fragments containing Ala-Pro in the right-handed alpha-helical conformation have been carried out using flexible geometry. The present calculations indicate that the right-handed alpha-helical region of conformational space is an energetically favored region that can also accommodate Ala-Pro in longer segments of right-handed alpha-helix. This is achieved due to minor variations in some of the internal parameters. Deviations in the backbone parameters of proline in the right-handed alpha-helix lead to a kink of about 23 degrees in the helix axis. These deviations have been characterized and a set of standard values has been suggested for producing such a kink. These values can be used for model building and as starting points for further minimization studies. Previous energy minimization studies have been done using rigid geometry. This may explain why the minimum for Ala-Pro in the right-handed alpha-helical region has not been recognized thus far.     

Positional preference of proline in alpha-helices.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.     

Structure of valinomycin by molecular dynamics studies.

Valinomycin is an important ionophore which exhibits a high conformational flexibility. The study of various conformations adopted by this molecule together with the study of flexibility in a given conformation can throw light on the ion transport by the ionophore across the membrane. Molecular dynamics (MD) studies are ideal to characterize the flexibility in different parts of the molecule and can also give an idea of various conformations adopted by the molecule at a given temperature. Hence MD studies at 100K have been carried out on the minimized crystal structure of the molecule to scan the possible conformations in the neighbourhood of the well known 'bracelet' like structure of uncomplexed Valinomycin, Properties, like the flexibility, average values, r.m.s. fluctuations of the various intramolecular hydrogen bonds are discussed. Energy minimization has been carried out on selected MD simulated points to analyze the characteristics of the unique conformation adopted by this molecule at this temperature.     

Energy of stabilization of the right-handed beta alpha beta crossover in proteins

An explanation in terms of conformational energies is provided for the observed nearly exclusive preference of the beta alpha beta structure for forming a right-handed, rather than a left-handed, crossover connection. Conformational energy computations have been carried out on a model beta alpha beta structure, consisting of two six-residue Val beta-strands and of a 12-residue Ala alpha-helix, connected by two flexible four-residue Ala links to the strands. The energy of the most favorable right-handed crossover is 15.51 kcal/mol lower than that of the corresponding left-handed cross-over. The right-handed crossover is a strain-free structure. Its energy of stabilization arises largely from the interactions of the two beta-strands with one another and with the alpha-helix. On the other hand, the left-handed crossover is either disrupted after energy minimization or it remains conformationally strained, as indicated by an energetically unfavorable left twisting of the beta-sheet and by the presence of high-energy local residue conformations. In the energetically most favorable right-handed crossover, the right twisting of the beta-sheet and its manner of interacting with the alpha-helix are identical with those computed earlier for isolated beta-sheets and for packed alpha/beta structures. This result supports a proposed principle that it is possible to account for the main features of frequently occurring structural arrangements in globular proteins in terms of the properties of their component structural elements.     

Role of hydrophobicity and solvent-mediated charge-charge interactions in stabilizing alpha-helices.

A theoretical study to identify the conformational preferences of lysine-based oligopeptides has been carried out. The solvation free energy and free energy of ionization of the oligopeptides have been calculated by using a fast multigrid boundary element method that considers the coupling between the conformation of the molecule and the ionization equilibria explicitly, at a given pH value. It has been found experimentally that isolated alanine and lysine residues have somewhat small intrinsic helix-forming tendencies; however, results from these simulations indicate that conformations containing right-handed alpha-helical turns are energetically favorable at low values of pH for lysine-based oligopeptides. Also, unusual patterns of interactions among lysine side chains with large hydrophobic contacts and close proximity (5-6 A) between charged NH3+ groups are observed. Similar arrangements of charged groups have been seen for lysine and arginine residues in experimentally determined structures of proteins available from the Protein Data Bank. The lowest-free-energy conformation of the sequence Ac-(LYS)6-NMe from these simulations showed large pKalpha shifts for some of the NH3+ groups of the lysine residues. Such large effects are not observed in the lowest-energy conformations of oligopeptide sequences with two, three, or four lysine residues. Calculations on the sequence Ac-LYS-(ALA)4-LYS-NMe also reveal low-energy alpha-helical conformations with interactions of one of the LYS side chains with the helix backbone in an arrangement quite similar to the one described recently by (Proc. Natl. Acad. Sci. U.S.A. 93:4025-4029). The results of this study provide a sound basis with which to discuss the nature of the interactions, such as hydrophobicity, charge-charge interaction, and solvent polarization effects, that stabilize right-handed alpha-helical conformations.     

Crystal structure and conformational analysis of ampullosporin A.

Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A.     

Folding study of an Aib-rich peptide in DMSO by molecular dynamics simulations.

To evaluate the ability of molecular dynamics (MD) simulations using atomic force-fields to correctly predict stable folded conformations of a peptide in solution, we show results from MD simulations of the reversible folding of an octapeptide rich in alpha-aminoisobutyric acid (2-amino-2-methyl-propanoic acid, Aib) solvated in di-methyl-sulfoxide (DMSO). This solvent generally prevents the formation of secondary structure, whereas Aib-rich peptides show a high propensity to form secondary structural elements, in particular 3(10)- and alpha-helical structures. Aib is, moreover, achiral, so that Aib-rich peptides can form left- or right-handed helices depending on the overall composition of the peptide, the temperature, and the solvation conditions. This makes the system an interesting case to study the ensembles of peptide conformations as a function of temperature by MD simulation. Simulations involving the folding and unfolding of the peptide were performed starting from two initial structures, a right-handed alpha-helical structure and an extended structure, at three temperatures, 298 K, 340 K, and 380 K, and the results are compared with experimental nuclear magnetic resonance (NMR) data measured at 298 K and 340 K. The simulations generally reproduce the available experimental nuclear Overhauser effect (NOE) data, even when a wide range of conformations is sampled at each temperature. The importance of adequate statistical sampling in order to reliably interpret the experimental data is discussed.     

Influence of solvent molecules on the stereochemical code of glycyl residues in proteins

The Ramachandran steric map and energy diagrams of the glycyl residue are symmetric. A plot of (phi,psi) angles of glycyl residues in 250 nonhomologous and high-resolution protein structures is also largely symmetric. However, there is a clear aberration in the symmetry. Although there is a cluster of points corresponding to the right-handed alpha-helical region, the "equivalent" cluster is clearly shifted to in and around the (phi,psi) values of (90 degrees, 0 degrees ) instead of being centered at the left-handed alpha-helical region of (60 degrees, 40 degrees ). This lack of symmetry exists even in the (phi,psi) distribution of residues from non-alpha-helical regions in proteins. Here we provide an explanation for this observation. An analysis of glycyl conformations in small peptide structures and in "coil" proteins, which are largely devoid of helical and sheet regions, shows that glycyl residues prefer to adopt conformations around (+/-90 degrees, 0 degrees ) instead of right- and left-handed alpha-helical regions. By using theoretical calculations, such conformations are shown to have highest solvent accessibility in a system of two-linked peptide units with glycyl residue at the central C(alpha) atom. This finding is consistent with the observations from 250 nonhomologous protein structures where glycyl residues with conformations close to (+/-90 degrees, 0 degrees ) are seen to have high solvent accessibility. Analysis of a subset of nonhomologous structures with very high resolution (1.5 A or better) shows that water molecules are indeed present at distances suitable for hydrogen bond interaction with glycyl residues possessing conformations close to (+/-90 degrees, 0 degrees ). It is suggested that water molecules play a key role in determining and stabilizing these conformations of glycyl residues and explain the aberration in the symmetry of glycyl conformations in proteins.     

Prediction of the three-dimensional structure of human growth hormone

In recent years, the protein-folding problem has attracted the attention of molecular biologists. Efforts have focused on developing heuristic and energy-based algorithms to predict the three-dimensional structure of a protein from its amino acid sequence. We have applied a series of heuristic algorithms to the sequence of human growth hormone. A family of five structures which are generically right-handed fourfold alpha-helical bundles are found from an investigation of approximately 10(8) structures. A plausible receptor binding site is suggested. Independent crystallographic analysis confirms some aspects of these predictions. These methods only deal with the "core" structure, and conformations of many residues are not defined. Further work is required to identify a unique set of coordinates and to clarify the topological alternative available to alpha-helical proteins.     

Characterization of proline-containing α-helix (helix F model of bacteriorhodopsin) by molecular dynamics studies

Many of the bilayer spanning segments of membrane transport proteins contain proline residues, and most of them are believed to occur in α-helical form. A proline residue in the middle of an α-helix is known to produce a bend in the helix, and recent studies have focused on characterizing such a bend at atomic level. In the present case, molecular dynamics (MD) studies are carried out on helix F model of bacteriorhodopsin (BR) Ace-(Ala)₇-Trp-(Ala)₂-Tyr-Pro-(Ala)₂-Trp-(Ala)₈-NHMe and compared with Ace-(Ala)₇-Trp-(Ala)₂-Tyr-(Ala)₃-Trp-(Ala)₈-NHMe in which the proline is replaced by alanine. The bend in the helix is characterized by structural parameters such as kink angle (α), wobble angle (θ), virtual torsion angle (ρ), and the hydrogen bond distance d (O_p?3 … N_p+1). The average values and the flexibility involved in these parameters are evaluated. The correlation among the bend related parameters are estimated. The equilibrium side chain orientations of tryptophan and tyrosine residues are discussed and compared with those found in the recently proposed model of bacteriorhodopsin. Finally, a detailed characterization of the bend in terms of secondary structures such as α_I, α_II and goniometric helices are discussed, which can be useful in the interpretation of the experimental results on the secondary structures of membrane proteins involving the proline residue. © 1993 Wiley-Liss, Inc.     

A structural and functional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid binding proteins

We have used NMR spectroscopy and limited proteolysis to characterize the structural properties of the Parkinson's disease-related protein alpha-synuclein in lipid and detergent micelle environments. We show that the lipid or micelle surface-bound portion of the molecule adopts a continuously helical structure with a single break. Modeling alphaS as an ideal alpha-helix reveals a hydrophobic surface that winds around the helix axis in a right-handed fashion. This feature is typical of 11-mer repeat containing sequences that adopt right-handed coiled coil conformations. In order to bind a flat or convex lipid surface, however, an unbroken helical alphaS structure would need to adopt an unusual, slightly unwound, alpha11/3 helix conformation (three complete turns per 11 residues). The break we observe in the alphaS helix may allow the protein to avoid this unusual conformation by adopting two shorter stretches of typical alpha-helical structure. However, a quantitative analysis suggests the possibility that the alpha11/3 conformation may in fact exist in lipid-bound alphaS. We discuss how structural features of helical 11-mer repeats could play a role in the reversible lipid binding function of alpha-synuclein and generalize this argument to include the 11-mer repeat-containing apolipoproteins, which also require the ability to release readily from lipid surfaces. A search of protein sequence databases confirms that synuclein-like 11-mer repeats are present in other proteins that bind lipids reversibly and predicts such a role for a number of hypothetical proteins of unknown function.     

Conformational interconversion in compstatin probed with molecular dynamics simulations

Compstatin is a 13-residue cyclic peptide that has the potential to become a therapeutic agent against unregulated complement activation. In our effort to understand the structural and dynamic characteristics of compstatin that form the basis for rational and combinatorial optimization of structure and activity, we performed 1-ns molecular dynamics (MD) simulations. We used as input in the MD simulations the ensemble of 21 lowest energy NMR structures, the average minimized structure, and a global optimization structure. At the end of the MD simulations we identified five conformations, with populations ranging between 9% and 44%. These conformations are as follows: 1) coil with alphaR-alphaR beta-turn, as was the conformation of the initial ensemble of NMR structures; 2) beta-hairpin with epsilon-alphaR beta-turn; 3) beta-hairpin with alphaR-alphaR beta-turn; 4) beta-hairpin with alphaR-beta beta-turn; and 5) alpha-helical. Conformational switch was possible with small amplitude backbone motions of the order of 0.1-0.4 A and free energy barrier crossing of 2-11 kcal/mol. All of the 21 MD structures corresponding to the NMR ensemble possessed a beta-turn, with 14 structures retaining the alphaR-alphaR beta-turn type, but the average minimized structure and the global optimization structures were converted to alpha-helical conformations. Overall, the MD simulations have aided to gain insight into the conformational space sampled by compstatin and have provided a measure of conformational interconversion. The calculated conformers will be useful as structural and possibly dynamic templates for optimization in the design of compstatin using structure-activity relations (SAR) or dynamics-activity relations (DAR).     

Molecular dynamics simulations of Ac-3Aib-Cage-3Aib-NHMe

Solvents play a stabilising role with the more stable conformations obtained in polar solvents than in vacuo. We investigate to what extent the structural propensities of the pentacyclo-undecane (PCU) cage polypeptide chain of the type Ac-3Aib-Cage-3Aib-NHMe are influenced in implicit water and in explicit solvents: methanol (MEOH), dimethyl sulphoxide (DMSO) and TIP3P water. The sampling of the α-helical conformations of the PCU cage polypeptide was investigated using the in-house modified PARM94 force-field parameters. Analysis of 50 ns molecular dynamics (MD) simulations revealed a tendency of the PCU cage polypeptide to assume bent structures, especially in polar solvents. The choice of solvents was designed to relate the simulations to physiological conditions. The individual amino-isobutyric acid residues predominantly sampled the right-handed and left-handed 3₁₀-helical conformations, indicating that the helical conformations are preferred in all four environments (in vacuo, MEOH, water and DMSO). Additionally, the 100 ns replica exchange MD (REMD) simulations of the PCU cage polypeptide in implicit water revealed more conformational variety present than in explicit solvents, and is more consistent with previous theoretical studies on the PCU cage residue. The present theoretical results may help in rationalising experimental results on these PCU cage polypeptides, and definitely show the importance of a dynamical approach for a correct interpretation and prediction of the conformational behaviour of the PCU cage molecules in different environments.     

Comparison of the conformation and electrostatic surface properties of magainin peptides bound to sodium dodecyl sulfate and dodecylphosphocholine micelles

The role played by noncovalent interactions in inducing a stable secondary structure onto the sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelle-bound conformations of (Ala(8,13,18))magainin 2 amide and the DPC micelle bound conformation of magainin 1 were determined. Two-dimensional NMR and molecular modeling investigations indicated that (Ala(8,13,18))magainin 2 amide bound to DPC micelles adopts a alpha-helical secondary structure involving residues 2-16. The four C-terminal residues converge to a lose beta-turn structure. (Ala(8,13,18))magainin 2 amide bound to SDS miscelles adopts a alpha-helical secondary structure involving residues 7-18. The C- and N-terminal residues exhibited a great deal of conformational flexibility. Magainin 1 bound to DPC micelles adopts a alpha-helical secondary structure involving residues 4-19. The C-terminal residues converge to a lose beta-turn structure. The results of this investigation indicate hydrophobic interactions are the major contributors to stabilizing the induced helical structure of the micelle-bound peptides. Electrostatic interactions between the polar head groups of the micelle and the cationic side chains of the peptides define the positions along the peptide backbone where the helical structures begin and end.     

Molecular mechanism of Ras-related protein Rab-5A and effect of mutations in the catalytically active phosphate-binding loop

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a was spotted in the cytoplasmic side of early endosomes in association with the GTP. Previous study suggested that the substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold, while, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a non-hydrolyzable GTP analog guanosine-5′-(β, γ)-imidotriphosphate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite an in silico approach, this study provides a deep insight into the impact of mutation on the structure, function, stability, and mechanism of binding of GppNHp to the Rab5a at molecular level.     

Conformational behavior of temporin A and temporin L in aqueous solution: a computational/experimental study

Molecular dynamics (MD) simulations and circular dichroism (CD) experiments were carried out on aqueous temporin A and L, two short peptides belonging to an interesting class of natural substances known to be active mainly against Gram-positive/negative bacteria and fungi. Experimental results indicate the higher propensity of temporin L, with respect to temporin A, in forming alpha-helical structures. These results were revisited by long-timescale MD simulations, in which their alpha-helical propensity was investigated in the absence of trifluoroethanol. Results clearly show the higher stability of alpha-helix conformations in temporin L; moreover, an interestingly strong mechanical analogy emerges since both temporins show the same residue interval (from 7 to 10) as the most energetically accessible for alpha-helix formation. Such studies provide some intriguing structural and mechanical evidence that may help in better understanding and rationalizing the conformational behaviour of temporins in water solution and, ultimately, the inner principles of their microbial targets selectivity and mechanism of action at the level of cell membranes.     

The planar conformation of a strained proline ring: a QM/MM study

QM and QM/MM energy calculations have been carried out on an atomic resolution structure of liganded triosephosphate isomerase (TIM) that has an active site proline (Pro168) in a planar conformation. The origin of the planarity of this proline has been identified. Steric interactions between the atoms of the proline ring and a tyrosine ring (Tyr166) on one side of the proline prevent the ring from adopting the up pucker (chi1 is approximately -30 degrees), while the side chain of a nearby alanine (Ala171) forbids the down pucker (chi1 is approximately +30 degrees). To obtain a proline conformation that is in agreement with the experimentally observed planar state, a quantum system of sufficient size is required and should at least include the nearby side chains of Tyr166, Ala171, and Glu129 to provide enough stabilization. It is argued that the current force fields for structure optimization do not describe strained protein fragments correctly. The proline is part of a catalytic loop that closes upon ligand binding. Comparison of the proline conformation in different TIM X-ray structures, indicates that in the closed conformation of TIM the proline is planar or nearly planar, while in the open conformation it is down puckered. This suggests that the planarity possibly plays a role in the overall catalytic cycle of TIM, presumable acting as a reservoir of energy that becomes available upon loop opening.     

Characteristics of distribution of amino acid residues in the primary structure of calmodulin

Systemic analysis of the peculiarities of distribution of di-, tri- and tetrapeptide residues in amino acid sequence of calmodulins of different origin has been carried out. A conclusion is made that all the examined repeated tri- and tetrapeptide residues with comparatively low conformation mobility enter the alpha-helical conformations with low mobility in beta-turns, and tri- and tetrapeptides with intermediate meaning of conformation entropy in beta-sheet conformations of the calmodulin molecule.     

The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites

A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.