The backrub motion: how protein backbone shrugs when a sidechain dances

Surprisingly, the frozen structures from ultra-high-resolution protein crystallography reveal a prevalent, but subtle, mode of local backbone motion coupled to much larger, two-state changes of sidechain conformation. This "backrub" motion provides an influential and common type of local plasticity in protein backbone. Concerted reorientation of two adjacent peptides swings the central sidechain perpendicular to the chain direction, changing accessible sidechain conformations while leaving flanking structure undisturbed. Alternate conformations in sub-1 angstroms crystal structures show backrub motions for two-thirds of the significant Cbeta shifts and 3% of the total residues in these proteins (126/3882), accompanied by two-state changes in sidechain rotamer. The Backrub modeling tool is effective in crystallographic rebuilding. For homology modeling or protein redesign, backrubs can provide realistic, small perturbations to rigid backbones. For large sidechain changes in protein dynamics or for single mutations, backrubs allow backbone accommodation while maintaining H bonds and ideal geometry.     

Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks

A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, ≥90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed (?, ψ) torsion angles of ca 12º. TALOS-N also reports sidechain χ¹ rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.     

Equilibrium transitions between side‐chain conformations in leucine and isoleucine

Despite recent improvements in computational methods for protein design, we still lack a quantitative, predictive understanding of the intrinsic probabilities for amino acids to adopt particular side‐chain conformations. Surprisingly, this question has remained unsettled for many years, in part because of inconsistent results from different experimental approaches. To explicitly determine the relative populations of different side‐chain dihedral angles, we performed all‐atom hard‐sphere Langevin Dynamics simulations of leucine (Leu) and isoleucine (Ile) dipeptide mimetics with stereo‐chemical constraints and repulsive‐only steric interactions between non‐bonded atoms. We determine the relative populations of the different χ₁ and χ₂ dihedral angle combinations as a function of the backbone dihedral angles ? and ψ. We also propose, and test, a mechanism for inter‐conversion between the different side‐chain conformations. Specifically, we discover that some of the transitions between side‐chain dihedral angle combinations are very frequent, whereas others are orders of magnitude less frequent, because they require rare coordinated motions to avoid steric clashes. For example, to transition between different values of χ₂, the Leu side‐chain bond angles κ₁ and κ₂ must increase, whereas to transition in χ₁, the Ile bond angles λ₁ and λ₂ must increase. These results emphasize the importance of computational approaches in stimulating further experimental studies of the conformations of side‐chains in proteins. Moreover, our studies emphasize the power of simple steric models to inform our understanding of protein structure, dynamics, and design. Proteins 2015; 83:1488–1499. © 2015 Wiley Periodicals, Inc.     

Analysing the ability to retain sidechain hydrogen-bonds in mutant proteins

MOTIVATION: Hydrogen bonds are one of the most important inter-atomic interactions in biology. Previous experimental, theoretical and bioinformatics analyses have shown that the hydrogen bonding potential of amino acids is generally satisfied and that buried unsatisfied hydrogen-bond-capable residues are destabilizing. When studying mutant proteins, or introducing mutations to residues involved in hydrogen bonding, one needs to know whether a hydrogen bond can be maintained. Our aim, therefore, was to develop a rapid method to evaluate whether a sidechain can form a hydrogen-bond. RESULTS: A novel knowledge-based approach was developed in which the conformations accessible to the residues involved are taken into account. Residues involved in hydrogen bonds in a set of high resolution crystal structures were analyzed and this analysis is then applied to a given protein. The program was applied to assess mutations in the tumour-suppressor protein, p53. This raised the number of distinct mutations identified as disrupting sidechain-sidechain hydrogen bonding from 181 in our previous analysis to 202 in this analysis.     

An experimental Ramachandran plot for retropeptide derivatives: Conformational features of derivatives of GEM-diamino and malonyl amino acids

Malonic and diaminomethane residues, equivalent to the two possible retro modifications of a glycine unit, with an inverted peptide group, present particular conformations that differ from those found in glycine and, in general, in α-amino acids. In both cases the φ_i and ψ_i torsional angles have restricted values as deduced from inspection of the Cambridge Structural Data Bank and from compounds studied by us. Thus, both ψ_i angles tend to be equal to 115° (or −115°) in the malonyl residues, whereas the φ_i angles tend to be equal to 88° (or −88°) in the diaminomethane residues. These results are in agreement with previous experimental data on polymers, but in the case of malonyl residues they differ from theoretical calculations on isolated molecules. The experimental data for both residues can be represented in a way similar to the usual Ramachandran plot, which will be useful in analyzing the incorporation of these residues into proteins. When side chains are present in either type of residue, they are similar to conventional α-amino acids, although the orientation of the peptide groups is different. In such cases they acquire conformations similar to those found in peptide residues in the α-helix and β-sheet conformations, although other conformations are also possible. © 1998 John Wiley & Sons, Inc. Biopoly 45: 149–155, 1998     

Comparative analysis of 13C chemical shifts of β-sheet amyloid proteins and outer membrane proteins

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the ¹³C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The ¹³C chemical shifts are shown in 2D ¹³C–¹³C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The ¹³C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

     

Stereochemistry of nucleic acids and their constituents. IV. Allowed and preferred conformations of nucleosides,nucleoside mono-, di-, tri-, tetraphosphates,nucleic acids and polynucleotides

A uniform notation and convention is suggested to describe the torsional angles in nucleic acids and their derivatives. The torsional angle χ, relating the stereochemistry of the base with respect to the sugar, shows more variation for the β-purine glycosides than for the β-pyrimidine glycosides. This variation is attributed to the fact that the β-purine derivatives may form intramolecular O(5′)-H…N(3) hydrogen bonding. The χ values for the α-purine and α-pyrimidine glycosides show preference for the –syn-clinal (or anti) conformation. The mode of puckering of the sugar also influences the χ value. The various possible conformations for the furanose ring are described by the torsional angles τ₀ τ₁, τ₂, τ₃, τ₄, about the five ring bonds. From an analysis of the torsional angles (ω, ?, ψ, ψ′, ?′, ω′) about the sugar phosphate bonds in the x-ray structures of the known nucleosides, nucleotides, phosphodiesters, nucleic acids, and related compounds, and from a consideration of molecular models, it is found that the possible conformations for the backbone of helical nucleic acids is strikingly limited. Most importantly, the preferred conformation of the nucleotide unit in poly nucleotides and nucleic acids turns out to be the same as that found for the nucleotide in the crystal structure. It is observed that base “stacking” is a consequence of the restricted backbone conformation. The torsional angles are illustrated in the form of conformational “wheels”. Interrelation between the torsion angles about successive pairs of sugar-phosphate bonds are presented in the form of conformational maps: ω,?; ?,ψ; ψ.ψ′; ψ′,?′; ?′,ω′; ω′,ω. The ω′,ω map shows the perferred conformations about the inter-nucleotide bonds of right- and left-handed helices and the possible conformations of phosphodiesters. The preferred conformation of the pyrophosphate and triphosphate is that in which the phosphate oxygens display a staggered arrangement when viewed along the P–P axis. A plausible structure and conformation for the ATPM^2? backbound complex is presented. This structure differs from that proposed by SzentGyorgi in that the metal (only transition metals are considered here) is not bound to the NH₂ nitrogen of adenine, but rather is simultaneously bound to N(7) of the ring and three phosphates (α, β, γ), or N(7) of the ring and two phosphates (β, γ). The remaining metal coordination may be satisfied by solvent–metal or enzyme–metal bonds.     

An evaluvation of discrete and continuum search techniques for conformational analysis of side chains in proteins

Methodology for calculation of side-chain conformations in proteins is evaluated. The role and impact of corrections to idealized rotameric structures are considered, by incorporating methods for torsional optimization into rotamer-packing algorithms. Off-rotamer corrections given by continuum torsional optimization improve, over simpler rotamer-packing procedures, the accuracy with which the conformations of side chains of buried amino acids can be predicted. The analogy between protein side-chain calculations and spin systems is explored by adapting spin simulation methods to side-chain packing algorithms. Implementations of mean-field and heat-bath algorithms for side-chain packing are described and their performance tested. The procedures introduced here address the combinatorial problem in an efficient and reasonably effective manner, as evidenced by analysis of their convergence properties. Application of refined protocols yields overall prediction accuracies of 80% for χ¹ and 68percnt; for χ^1,2 pairs for a test set of 60 proteins, using a 40° cutoff to define correct placement. For buried amino acids (defined as having less than 30% relative solvent accessibility) the prediction accuracies increase to 88percnt; for χ¹ and 79percnt; for χ^1,2 pairs. The influence of the form of the potential energy function is studied by comparing results obtained with 12-6 and 9-6 potentials. The 9-6 form leads to more accurate results. Detailed comparison with previous work is presented, and the effect of combinatorial packing steps is shown to be important for all but the smallest proteins. © 1995 John Wiley & Sons, Inc.     

Effect of hydrophobic core packing on sidechain dynamics

The effect of hydrophobic core packing on sidechain dynamics was analyzed by comparing the dynamics of wild-type (WT) ubiquitin to those of a variant which has seven core mutations. This variant, 1D7, was designed to resemble WT by having a well-packed core of similar volume, and we find that its overall level of dynamics is only subtly different from WT. However, the mutations caused a redistribution in the positions of core residues that are dynamic. This correlates with the tendency of these residues to populate unfavorable rotamers, suggesting that strain from poor sidechain conformations may promote increased flexibility as a mechanism to relieve unfavorable steric interactions. The results demonstrate that even when core volume is conserved, different packing arrangements in mutants can alter dynamic behavior.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

Accuracy of side-chain prediction upon near-native protein backbones generated by ab initio folding methods

The ab initio folding problem can be divided into two sequential tasks of approximately equal computational complexity: the generation of native-like backbone folds and the positioning of side chains upon these backbones. The prediction of side-chain conformation in this context is challenging, because at best only the near-native global fold of the protein is known. To test the effect of displacements in the protein backbones on side-chain prediction for folds generated ab initio, sets of near-native backbones (≤ 4 Å Cα RMS error) for four small proteins were generated by two methods. The steric environment surrounding each residue was probed by placing the side chains in the native conformation on each of these decoys, followed by torsion-space optimization to remove steric clashes on a rigid backbone. We observe that on average 40% of the χ1 angles were displaced by 40° or more, effectively setting the limits in accuracy for side-chain modeling under these conditions. Three different algorithms were subsequently used for prediction of side-chain conformation. The average prediction accuracy for the three methods was remarkably similar: 49% to 51% of the χ1 angles were predicted correctly overall (33% to 36% of the χ1+2 angles). Interestingly, when the inter-side-chain interactions were disregarded, the mean accuracy increased. A consensus approach is described, in which side-chain conformations are defined based on the most frequently predicted χ angles for a given method upon each set of near-native backbones. We find that consensus modeling, which de facto includes backbone flexibility, improves side-chain prediction: χ1 accuracy improved to 51–54% (36–42% of χ1+2). Implications of a consensus method for ab initio protein structure prediction are discussed. Proteins 33:204–217, 1998. © 1998 Wiley-Liss, Inc.     

Conformational preferences of amino acid side chains in collagen

Conformational energy computations were carried out on collagenlike triple-stranded conformations of several poly(tripeptide)s with the general structure CH₃CO? (Gly? X? Y)₃? NHCH₃. The sequences considered had various amino acid residues in position X or Y of the central tripeptide, with either Pro or Ala as a neighbor, i.e., Gly-X-Pro, Gly-X-Ala, Gly-Pro-Y, and Gly-Ala-Y. Minimum-energy conformations were computed for the side chains, and their distributions were compared for the four sequences. The residues used were Abu (= α-aminobutyric acid), Leu, Phe, Ser, Asp, Asn, Val, Ile, and Thr. The conformational energy of a ? Ch₂? CH₃ side chain in Abu was mapped as a function of the dihedral angle χ¹. Intrastrand interactions with neighboring residues do not affect the conformations of a side chain in position Y, and they have a minor effect on it in the X-Ala sequence, but they strongly restrict the conformational freedom of the side chain in the X-Pro sequence. Conversely, interstrand interactions do not affect side chains in position X, but they strongly restrict the conformational freedom of a side chain in position Y if there is a nearby Pro residue in a neighboring strand. Hydrogen bonds with the backbone can be formed in some conformations of long polar side chains, such as Asp, Asn, or Gln. All amino acid residues can be accommodated in collagen. Because of the interactions mentioned above, steric and energetic constraints can be correlated with observed preferences of certain amino acids for positions X or Y in collagen. Hence, these preferences may be explained, in part, in terms of differences in the conformational freedom of the side chains in the triple-stranded structure.     

Analysis of side-chain orientations in homologous proteins

The side-chain conformations of topologically equivalent residues in seven pairs of proteins ranging in sequence homology from 16% to 60% are compared. Both identical and mutated residues are included. For proteins with greater than 40% homology, it is found that at least 80% of the side-chain orientations of identical residues and 75% or more of the mutated residues in each pair of proteins have matching gamma atom dihedral angles (+/- 40 degrees); the comparison is not based strictly on chi 1 angles. Further, if a match is obtained at the gamma position, there is a high probability of matching for the delta atom(s) of the side-chain. For proteins with less than 25% homology the percentages are somewhat lower. Trends observed for conservative substitutions are essentially the same as those noted for mutated residues in general. Side-chain accessibility does not affect the probability of matches of identical residues; however, less accessible pairs of mutated residues have 10 to 20% higher matching probabilities than do exposed residues. Mismatches can frequently be related to large B-factors, certain types of amino acid substitutions, or the appearance of multiple minima on the side-chain potential energy surfaces and are most likely to occur for certain small residues (Ser, Thr, Val). Analysis of all the results makes possible the formulation of a set of rules for side-chain positioning in the modeling of homologous proteins.     

Calibration of effective van der Waals atomic contact radii for proteins and peptides

Effective van der Waals radii were calibrated in such a way that molecular models built from standard bond lengths and bond angles reproduced the amino acid conformations observed by crystallography in proteins and peptides. The calibrations were based on the comparison of the Ramachandran plots prepared from high-resolution X-ray data of proteins and peptides with the allowed phi, psi torsional angle space for the dipeptide molecular models. The calibrated radii are useful as criteria with which to filter energetically improbable conformations in molecular modeling studies of proteins and peptides.     

Studies on the conformation of amino acids. X. Conformations of norvalyl, leucyl and aromatic side groups in a dipeptide unit

Backbone-side group conformations of amino acid residues including one or two δ-carbons in the side group have been investigated. Conformational energies of norvalyl, leucyl, phenylalanyl, tyrosyl, tryptophenyl, and histidinyl side groups in a dipeptide unit have been calculated by using classical energy expressions. The side group conformations about the C^α—C^β and C^β—C^γ bonds are restricted to specific values of the respective rotational angles. Thus, most favourable positions of γ- and δ-atoms of a linear side-chain (norvalyl) are restricted to (γ_I, δ_II) (γ_II, δ_I), (γ_II, δ_II), (γ_III, δ_II), and (γ_III, δ_III), whereas those of the side-chain branching at a sp³ γ-atom (leueyl) are further restricted. It is also shown that there is a definite correlation between the orientations of the two peptide planes and that of the planar group of the aromatic side chain of phenylalanyl type residues. The studies bring out an important fact that while the γ-atoms have definite and characteristic effects on the backbone rotational angles ? and ψ, the δ atoms and beyond have no effects on the preferred ? and ψ values. Thus, the preferred backbone conformations are independent of the preferred side group conformations beyond the γ-atom and vice versa. The observed ?, ψ, χ¹, and χ² values of amino acids, simple peptides, and of the three protein molecules lysozyme, myoglobin, and chymotrypsin have been compared with the theoretical predictions, and the agreement is found to be excellent.     

Modeling protein loops using a phi i + 1, psi i dimer database.

We present an automated method for modeling backbones of protein loops. The method samples a database of phi i + 1 and psi i angles constructed from a nonredundant version of the Protein Data Bank (PDB). The dihedral angles phi i + 1 and psi i completely define the backbone conformation of a dimer when standard bond lengths, bond angles, and a trans planar peptide configuration are used. For the 400 possible dimers resulting from 20 natural amino acids, a list of allowed phi i + 1, psi i pairs for each dimer is created by pooling all such pairs from the loop segments of each protein in the nonredundant version of the PDB. Starting from the N-terminus of the loop sequence, conformations are generated by assigning randomly selected pairs of phi i + 1, psi i for each dimer from the respective pool using standard bond lengths, bond angles, and a trans peptide configuration. We use this database to simulate protein loops of lengths varying from 5 to 11 amino acids in five proteins of known three-dimensional structures. Typically, 10,000-50,000 models are simulated for each protein loop and are evaluated for stereochemical consistency. Depending on the length and sequence of a given loop, 50-80% of the models generated have no stereochemical strain in the backbone atoms. We demonstrate that, when simulated loops are extended to include flanking residues from homologous segments, only very few loops from an ensemble of sterically allowed conformations orient the flanking segments consistent with the protein topology. The presence of near-native backbone conformations for loops from five different proteins suggests the completeness of the dimeric database for use in modeling loops of homologous proteins. Here, we take advantage of this observation to design a method that filters near-native loop conformations from an ensemble of sterically allowed conformations. We demonstrate that our method eliminates the need for a loop-closure algorithm and hence allows for the use of topological constraints of the homologous proteins or disulfide constraints to filter near-native loop conformations.     

Simulations of the static and dynamic molecular conformations of xyloglucan. The role of the fucosylated sidechain in surface-specific sidechain folding.

The hemicellulosic polysaccharide xyloglucan binds with a strong affinity to cellulosic cell wall microfibrils, the resulting heterogeneous network constituting up to 50% of the dry weight of the cell wall in dicotyledonous plants. To elucidate the molecular details of this interaction, we have performed theoretical potential energy calculations of the static and dynamic equilibrium conformations of xyloglucan using the GEGOP software. In particular, we have evaluated the preferred sidechain conformations of hexa-, octa-, deca- and heptadecasaccharide model fragments of xyloglucan for molecules with a cellulose-like, flat, glucan backbone, and a cellobiose-like, twisted, glucan backbone conformation. For the flat backbone conformation the determination of static equilibrium molecular conformations revealed a tendency for sidechains to fold onto one surface of the backbone, defined here as the H1S face, in the fucosylated region of the polymer. This folding produces a molecule that is sterically accessible on the opposite face of the backbone, the H4S face. Typically, this folding onto the H1S surface is significantly stabilized by favorable interactions between the fucosylated, trisaccharide sidechain and the backbone, with some stabilization from adjacent terminal xylosyl sidechains. In contrast, the trisaccharide sidechain folds onto the H4S face of xyloglucan fragments with a twisted backbone conformation. Preliminary NMR data on nonasaccharide fragments isolated from sycamore suspension-cultured cell walls are consistent with the hypothesis that the twisted conformation of xyloglucan represents the solution form of this molecule. Metropolis Monte Carlo (MMC) simulations were employed to assess sidechain flexibility of the heptadecasaccharide fragments. Simulations performed on the flat, rigid, backbone xyloglucan indicate that the trisaccharide sidechain is less mobile than the terminal xylosyl sidechains. MMC calculations on a fully relaxed molecule revealed a positive correlation between a specific trisaccharide sidechain orientation and the 'flatness' of the backbone glucosyl residues adjacent to this sidechain. These results suggest that the trisaccharide sidechain may play a role in the formation of nucleation sites that initiate the binding of these regions to cellulose. Based on these conformational preferences we suggest the following model for the binding of xyloglucan to cellulose. Nucleation of a binding site is initiated by the fucosylated, trisaccharide sidechain that flattens out an adjacent region of the xyloglucan backbone. Upon contacting a cellulose microfibril this region spreads by step-wise flattening of successive segments of the backbone. Self-association of xyloglucan molecules in solution may be prevented by the low frequency of formation of these nucleation sites and the geometry of the molecules in solution.     

Determination of the local structure of the protein insectotoxin I5A from the scorpion Buthus eupeus from 1H-NMR spectroscopy data

The local structure (torsion angles phi, psi and chi 1 of amino acid residues) of insectotoxin I5A (35 residues) of scorpion Buthus eupeus has been determined from cross-peak integral intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra and spin coupling constants of vicinal H--NC alpha--H and H--C alpha C beta--H protons. The local structure determination was carried out by fitting complete relaxation matrix of peptide unit protons (protons of a given residue and NH proton of the next residue in the amino acid sequence) with experimental NOESY cross-peak intensities. The obtained intervals of backbone torsional angles phi and psi consistent with NMR data were determined for all but Gly residues. The predominant C alpha--C beta rotamer of the side chain has been unambiguously determined for 42% of the insectotoxin amino acid residues whereas for another 46% residues experimental data are fitted equally well with two rotamers. Stereospecific assignments were obtained for 38% of beta-methylene groups. The determined torsional angles phi, psi and chi 1 correspond to the sterically allowed conformations of the amino acid residues and agree with the insectotoxin secondary structure established earlier by 1H NMR spectroscopy.     

PM3 and AM1 Study on β-N-acetyl-muramic Acid and 3 Murein Related Derivatives

The energetically favoured conformations of -N-acetyl-Muramic acid, its C6-O-acetylated form, the methylamide and the methyl-glycoside have been investigated using the semiempirical PM3 and AM1 methods. All these compounds are either components or fragmentary structures of the murein network. The atomic coordinates of the starting set of the -N-acetyl-Muramic acid molecule have been obtained by a PM3 minimization of one saccharide molecule cut out from the murein single strand model proposed by Barnickel at al. [1]. The sidegroups of the derivatives have been introduced by a molecular editor. These conformations served as starting points in conformational space for a grid search by scanning all sidechain torsional angles for non-hydrogen atoms with exception of the N-acetyl group which was held in cisoid position (i.e. N2-H bond is parallel to C1-H and C3-H bond) and only minimized. The PM3 method with an additional amide correction potential and the AM1 method were used. The torsional angle distributions of the lactyl sidechain (free acid and methylamide), the C6-O-acetylated sidechain and the C1-methoxy sidechain have been investigated, showing distinct energetically favoured torsional angle regions. The results are compared to earlier studies on -N-acetyl-Muramic acid by J.S. Yadav et al. [2,3] who were using the MNDO and PCILO methods and by P.N.S. Yadav et al. [4] who were using the empirical MM2 force-field.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020190     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1-25 was compared with that of a truncated homodimer (dSP-B_8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm^− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B_1-25 dimer. In contrast, the truncated dimer dSP-B_8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm^− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.