A Hydrogen Bonded Chain in Bactereorhodopsin by Computer Modelling Approach

Abstract

The seven α-helical segments of Bacteriorhodopsin (BR) passing through the membrane are investigated for a continuous Hydrogen Bonded Chain (HBC). The study is carried out by computer modelling approach. It is assumed that the seven helices are placed as (AGFEDCB), which has been accepted as the best model by several groups. Helices A, D, E and G are considered to be present in right handed α-helical conformation. The inter-orientation of these helices are represented by Eulerian angles α, β and γ. For the helices B, C and F which contain Proline in the middle, several conformational possibilities were considered. In these cases apart from the Eulerian angles α, β and γ, the dihedral angles φ_{p_1} and ψ_{p_1} of the residues that are succeeded by Proline residue in the helical regions were also used in fixing the position of the helices with respect to each other. All these parameters were varied to fit with the top, middle and bottom distances reported by electron diffraction studies. Good fit was obtained for all right handed α-helical conformations and also for helices B, C and F with a left handed turn at the residue preceeding proline. Hence two structures were analysed for continuous HBC, Structure I which contained all the seven helices in right handed α-helical conformation and Structure II, which had the helices A, D, E and G in right handed conformation and the helices B, C and F in right handed α-helical conformation with a left handed turn at the residue preceeding proline. All possible staggered conformations were considered for the side chains and the inter atomic distances were analysed for Hydrogen bonds. It was possible to obtain a continuous chain in both the structures which includes most of the residues found to be important by the experiments. However Lys-216 has to be considered in two different conformations to connect the cytoplasmic side with the extra cellular side. The overall height spanned by HBC is about 25Å. The chains obtained by both the structures I and II are analysed in terms of the conformational parameters. It has also been possible to place the retinal in the region as predicted by the experiments. The Tryptophan residues which affect the spectral characterestics can be aligned on either side of the retinal.     

Directional Information Transfer in Protein Helices

IN the course of studies on the relation between conformation and primary sequence in globular proteins, it has become clear that the choice a residue makes between a right handed α-helical conformation (H?) and any other conformation (H) is determined mainly by that residue and its near neighbours in the primary sequence^1–9. When looking for physical mechanisms to explain these findings it is important to know whether the influence of one residue on the conformational state of a neighbouring residue has any directional characteristics. The possibility of certain residues exerting helix-forming influence in either the COOH-terminal or NH₂-terminal direction preferentially is suggested by the non-random distribution of amino-acid residues between the two ends of helical regions in globular proteins^4,10. Ptitsyn's analysis⁴ suggested that a group of residues containing alanine and leucine tends to occur within helical regions, while positively and negatively charged side chain residues are distributed preferentially at the carboxyl-and amino-terminal ends, respectively, of helices.     

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.     

Conformation of di-n-propylglycine residues (Dpg) in peptides: crystal structures of a type I' beta-turn forming tetrapeptide and an alpha-helical tetradecapeptide.

The crystal structures of two oligopeptides containing di-n-propylglycine (Dpg) residues, Boc-Gly-Dpg-Gly-Leu-OMe (1) and Boc-Val-Ala-Leu-Dpg-Val-Ala-Leu-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe (2) are presented. Peptide 1 adopts a type I'beta-turn conformation with Dpg(2)-Gly(3) at the corner positions. The 14-residue peptide 2 crystallizes with two molecules in the asymmetric unit, both of which adopt alpha-helical conformations stabilized by 11 successive 5 --> 1 hydrogen bonds. In addition, a single 4 --> 1 hydrogen bond is also observed at the N-terminus. All five Dpg residues adopt backbone torsion angles (phi, psi) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-C(alpha)-C' (tau) and the observed backbone phi,psi values. For tau > 106 degrees, helices are observed, while fully extended structures are characterized by tau < 106 degrees. The mean tau values for extended and folded conformations for the Dpg residue are 103.6 degrees +/- 1.7 degrees and 109.9 degrees +/- 2.6 degrees, respectively.     

Local propensities and statistical potentials of backbone dihedral angles in proteins

The following three issues concerning the backbone dihedral angles of protein structures are presented. (1) How do the dihedral angles of the 20 amino acids depend on the identity and conformation of their nearest residues? (2) To what extent are the native dihedral angles determined by local (dihedral) potentials? (3) How to build a knowledge-based potential for a residue's dihedral angles, considering the identity and conformation of its nearest residues? We find that the dihedral angle distribution for a residue can significantly depend on the identity and conformation of its adjacent residues. These correlations are in sharp contrast to the Flory isolated-pair hypothesis. Statistical potentials are built for all combinations of residue triplets and depend on the dihedral angles between consecutive residues. First, a low-resolution potential is obtained, which only differentiates between the main populated basins in the dihedral angle density plots. Minimization of the dihedral potential for 125 test proteins reveals that most native alpha-helical residues (89%) and a large fraction of native beta-sheet residues (47%) adopt conformations close to their native one. For native loop residues, the percentage is 48%. It is also found that this fraction is higher for residues away from the ends of alpha or beta secondary structure elements. In addition, a higher resolution potential is built as a function of dihedral angles by a smoothing procedure and continuous functions interpolations. Monte Carlo energy minimization with this potential results in a lower fraction for native beta-sheet residues. Nevertheless, because of the higher flexibility and entropy of beta structures, they could be preferred under the influence of non-local interactions. In general, most alpha-helices and many beta-sheets are strongly determined by the local potential, while the conformations in loops and near the end of beta-sheets are more influenced by non-local interactions.     

Analysis of the code relating sequence to conformation in globular proteins. An informational analysis of the role of the residue in determining the conformation of its neighbours in the primary sequence

1. The effect exerted by a residue on the conformation of neighbouring residues was analysed by using data from nine globular proteins of known sequence and conformation. 2. An information measure was used which estimated the role of a residue in influencing neighbouring conformations and also its tendency to influence the lengths of runs of residues in that conformation. This measure was estimated for each residue in all conformations defined by domains on the varphi, psi diagram. 3. Plots of the information measure yielded an intercept, which was a measure of intra-residue information for a residue. The slope was a measure of the statistical co-operativity or tendency of the residue to influence the occurrence of its neighbours in runs of a particular conformation. Both parameters are a function of the residue type. Statistical co-operativity is found in the alpha(1)-helical (H(1)) and beta-pleated-sheet (P(2)) conformations and, to a lesser extent, in their distorted variants H(2) and P(1). 4. The directional nature of these influences for H(1) and P(2) conformations is illustrated by plots of the information measure against the distance m from the residue, for m=-10 to +10. 5. The results for statistical co-operativity are discussed in relation to theories of helix-coil and pleated-sheet-coil transitions. The value of the information-theory-derived parameters in obtaining s parameters for the Zimm & Bragg (1959) equations is illustrated. 6. Directional effects are discussed with particular relation to mechanisms of the termination of helices and the involvement of the alpha(II) conformation and also to discontinuities in pleated-sheet conformations.     

Effect of side chains on the conformational energy and rotational strength of the n-pi transition for some alpha-helical poly-alpha-amino acids

Calculations of the dependence of the conformational energy and the rotational strength of the amide n–π* electronic transition (in a series of α-helical polyhel-α- amino acids with different side chains) on conformation have been carried out. The conformational energies were computed by procedures developed in this laboratory; the computation of rotational strengths was carried out by the method of Schellman and Oriel, with a slight modification. Polyamino acids with both nonpolar and polar side chains were considered; in the latter case, it was assumed that the only influence of the polar side chain was on the backbone conformation and on the electrostatic field which perturbs the amide chromophore of the backbone. Only conformations in the range of backbone dihedral angles of the right- and left-handed a-helices were considered, and the assumption of regularity (i.e., uniformity of dihedral angles in every residue) was made. The rotational strength per residue was found to vary markedly with chain length (in oligomers of up to 40 residues long); both the conformational energy per residue and the rotational strength per residue were found to vary significantly with the backbone conformation, which in turn depends on the nature of the side chain. The geometry of the hydrogen bond in the α-helical backbone is the most important factor which influences the dependence of the rotational strength on conformation. The implications of these results, for the interpretation of experimental circular dichroism and optical rotatory dispersion data, are discussed.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Analysis of forces that determine helix formation in alpha-proteins

A model for prediction of alpha-helical regions in amino acid sequences has been tested on the mainly-alpha protein structure class. The modeling represents the construction of a continuous hypothetical alpha-helical conformation for the whole protein chain, and was performed using molecular mechanics tools. The positive prediction of alpha-helical and non-alpha-helical pentapeptide fragments of the proteins is 79%. The model considers only local interactions in the polypeptide chain without the influence of the tertiary structure. It was shown that the local interaction defines the alpha-helical conformation for 85% of the native alpha-helical regions. The relative energy contributions to the energy of the model were analyzed with the finding that the van der Waals component determines the formation of alpha-helices. Hydrogen bonds remain at constant energy independently whether alpha-helix or non-alpha-helix occurs in the native protein, and do not determine the location of helical regions. In contrast to existing methods, this approach additionally permits the prediction of conformations of side chains. The model suggests the correct values for ~60% of all chi-angles of alpha-helical residues.     

Aib residues in peptaibiotics and synthetic sequences: analysis of nonhelical conformations

The alpha-aminoisobutyric (Aib) residue has generally been considered to be a strongly helicogenic residue as evidenced by its ability to promote helical folding in synthetic and natural sequences. Crystal structures of several peptide natural products, peptaibols, have revealed predominantly helical conformations, despite the presence of multiple helix-breaking Pro or Hyp residues. Survey of synthetic Aib-containing peptides shows a preponderance of 3(10)-, alpha-, and mixed 3(10)/alpha-helical structures. This review highlights the examples of Aib residues observed in nonhelical conformations, which fall 'primarily' into the polyproline II (P(II)) and fully extended regions of conformational space. The achiral Aib residue can adopt both left (alpha(L))- and right (alpha(R))-handed helical conformations. In sequences containing chiral amino acids, helix termination can occur by means of chiral reversal at an Aib residue, resulting in formation of a Schellman motif. Examples of Aib residues in unusual conformations are illustrated by surveying a database of Aib-containing crystal structures.     

The role of PII conformations in the calculation of peptide fractional helix content.

Changes in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non-alpha-helical peptide acetylYEAAAKEAPAKEAAAKAamide generate changes in its dichroic spectrum characteristic for a conformational transition. This transition has the characteristic features of a residue PII/unstructured conformational equilibrium in which PII denotes an extended left-handed helical conformation and unstructured denotes all the remaining conformations in a random coil ensemble. Replacement of the proline residue facilitates population of residues in an alpha-helical conformation. However, the ellipticity values for these non-proline peptides merge with the ellipticity of the proline peptide as the population of residues in the alpha-helix conformation is diminished. This convergence suggests that all residues in a host/guest peptide series of the same length share a common PII/unstructured conformational equilibrium in a given solvent. We propose that the fractional helix content of peptides within such a series may be estimated by using a two-state calculation in which the ellipticity for the non-alpha-helix conformations is provided by a peptide having a central proline guest residue.     

Empirical evaluation of the influence of side chains on the conformational entropy of the polypeptide backbone

Changes in amino acid side chains have long been recognized to alterthe range and distribution of ?, ψ angles found in the main chain of polypeptides. Altering the range and distribution of ?, ψ angles also alters the conformational entropy of the flexible denatured state and may thus stabilize or destabilize it relative to the comparatively conformationally rigid native state. A database of 12,320 residues from 61 nonhomologous, high resolution crystal structures was examined to determine the ?, ψ conformational preferences of each of the 20 amino acids. These observed distributions in the native state of proteins are assumed to also reflect the distributions found in the denatured state. The distributionswere used to approximate the energy surface for each residue, allowing the calculation of relative conformational entropies for each residue relative to glycine. In the most extreme case, replacement of glycine by proline, conformational entropy changes will stabilize the native state relative to the denatured state by ?0.82 ± 0.08 kcal/mol at 20°C. Surprisingly, alanine is found to be the most ordered residue other than proline. This unexpected result is a result of the high percentage of alanines found in helical conformations. This either indicates that the observed distributions in the native state do not reflect the distributions in the denatured state, or that alanine is much more likely to adopt a helical conformation in the denatured state than residues with longer side chains. Among those residues with ?, ψ angles compatible with helix incorporation the percentage of alanines actually in helices is very similar to other residues. This and the consistent ordering of alanine relative to other residues regardless of secondary structure are evidence that ?, ψ distributions in native states reflect those in the denatured states. © 1995 Wiley-Liss, Inc.     

Proline-induced constraints in alpha-helices

The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala₄-Pro-NHMe and of Ac-Ala₇-Pro-Ala₇-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ? and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.     

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within a membrane protein sequence. However, these methods tend to disagree over the beginning and end residues of TM helices, posing problems for subsequent modeling and simulation studies. Molecular dynamics (MD) simulations in an explicit lipid and water environment are used to help define the TM helix of the M2 protein from influenza A virus. Based on a comparison of the results of five different secondary structure prediction algorithms, three different helix lengths (an 18mer, a 26mer, and a 34mer) were simulated. Each simulation system contained 127 POPC molecules plus approximately 3500-4700 waters, giving a total of approximately 18,000-21,000 atoms. Two simulations, each of 2 ns duration, were run for the 18mer and 26mer, and five separate simulations were run for the 34mer, using different starting models generated by restrained in vacuo MD simulations. The total simulation time amounted to 11 ns. Analysis of the time-dependent secondary structure of the TM segments was used to define the regions that adopted a stable alpha-helical conformation throughout the simulation. This analysis indicates a core TM region of approximately 20 residues (from residue 22 to residue 43) that remained in an alpha-helical conformation. Analysis of atomic density profiles suggested that the 18mer helix revealed a local perturbation of the lipid bilayer. Polar side chains on either side of this region form relatively long-lived H-bonds to lipid headgroups and water molecules.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Conformations of A-DNA and B-DNA in agreement with fiber X-ray and infrared dichroism

Two right handed double helices are proposed as models for DNA fibers in respectively the A and the B form. The present conformations have geometrical parameters which agree very well with fiber X-ray and the orientation of their phosphate groups is in good accordance with infrared dichroïsm measurements. Dihedral angles and complete sets of atomic coordinates are given together with stereo-views and curves of the calculated diffracted intensities. Results are compared with experimental data and with preceding DNA models.     

Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid-state NMR

There are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure-property relationship of fibrous proteins. Silk fibroin from a wild silkworm, Samia cynthia ricini, mainly consists of the repeated similar sequences by about 100 times where there are alternative appearances of the polyalanine (Ala)(12-13) region and the Gly-rich region. In this paper, a sequential model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical sequence of the silk fibroin, was synthesized, and the atomic-level conformations of Gly residues at the N- and C-terminal ends of the polyalanine region were determined as well as that of the central Ala residue using (13)C 2D spin diffusion solid-state nuclear magnetic resonance (NMR) under off-magic angle spinning. In the model peptide with alpha-helical conformation, the torsion angle of the central Ala residue, the 19th Ala, was determined to be (phi, psi) = (-60 degrees, -50 degrees ), which was a typical alpha-helical structure, but the torsion angles of two Gly residues, the 12th and 25th Gly residues, which are located at the N- and C-terminal ends of the polyalanine region, were determined to be (phi, psi) = (-70 degrees, -30 degrees ) and (phi, psi) = (-70 degrees, -20 degrees ), respectively. Thus, it was observed that the turns at both ends of polyalanine with alpha-helix conformation in the model peptide are tightly wound.     

Conformational and sequence signatures in beta helix proteins

beta helix proteins are characterized by a repetitive fold, in which the repeating unit is a beta-helical coil formed by three strand segments linked by three loop segments. Using a data set of left- and right-handed beta helix proteins, we have examined conformational features at equivalent positions in successive coils. This has provided insights into the conformational rules that the proteins employ to fold into beta helices. Left-handed beta helices attain their equilateral prism fold by incorporating "corners" with the conformational sequence P(II)-P(II)-alpha(L)-P(II), which imposes sequence restrictions, resulting in the first and third P(II) residues often being G and a small, uncharged residue (V, A, S, T, C), respectively. Right-handed beta helices feature mid-sized loops (4, 5, or 6 residues) of conserved conformation, but not of conserved sequence; they also display an alpha-helical residue at the C-terminal end of L2 loops. Backbone conformational parameters (phi,psi) that permit the formation of continuous, loopless beta helices (Perutz nanotubes) have also been investigated.     

The effects of changes in ring geometry on computer models of amylose

A review of single-crystal studies shows that α-D-glucopyranose, residues of which constitute the monomeric units of amylose, is flexible within the constraints of the Cl conformation, and that the internal differences among the rings are most clearly indicated by the variety in ring-torsion angles (or conformation angles). An index of the cumulative effect of changes in these angles is provided by the length of the virtual bond, O-1—O-4, and classification of residue geometries by virtual bond-length permits a systematic selection of suitable residues for the construction of models of amylose. By the use of D-glucopyranose residues having different geometries, it is possible to build models of (a) V-amylose helices having 6, 7, and 8 residues per turn, (b) single and double helical B-amyloses, and (c) KBr-amylose, all of which satisfy reasonable stereochemical criteria. Because no single residue can satisfactorily model all of the well known polymorphs of amylose, it is suggested that structural determinations that utilize a rigid residue approximation should make use of the full range of known, residue geometries.     

Structural analysis of peptide helices containing centrally positioned lactic acid residues

The effect of insertion of lactic acid (Lac) residues into peptide helices has been probed using specifically designed sequences. The crystal structures of 11-residue and 14-residue depsipeptides Boc-Val-Val-Ala-Leu-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (1) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (3), containing centrally positioned Lac residues, have been determined. The structure of an 11-residue peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (2), analog of a which is an amide previously determined Lac-containing depsipeptide, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe I. L. Karle, C. Das, and P. Balaram, Biopolymers, Vol. 59, (2001) pp. 276-289], is also reported. Peptide 1 adopts a helical fold, which is stabilized by mixture of 4-->1 and 5-->1 hydrogen bonds. Peptide 2 adopts a completely alpha-helical conformation stabilized by eight successive 5-->1 hydrogen bonds. Peptide 3 appears to be predominately alpha-helical, with seven 5-->1 hydrogen bonds and three 4-->1 interaction interspersed in the sequence. In the structure of peptide 3 in addition to water molecules in the head-to-tail region, hydration at an internal segment of the helix is also observed. A comparison of five related peptide helices, containing a single Lac residue, reveals that the hydroxy acid can be comfortably accommodated at interior positions in the helix, with the closest C=O...O distances lying between 2.8 and 3.3 A.