Characteristic sequential residue environment of amino acids in proteins

The occurrence of all di- and tripeptide segments of proteins was counted in a large data base containing about 119 000 residues. It was found that the abundance of the amino acids does not determine the frequency of the various di- and tripeptide segments. In addition, the frequency of the various tripeptides cannot be predicted from the observed pair-frequency values. The pair-frequency distribution of amino acids is highly asymmetrical, pairs formed from identical residues are generally preferred and amino acids cannot be clustered on the basis of their first neighbour preferences. These data indicate the existence of general short range regularities in the primary structure of proteins. The consequences of these short range regularities were studied by comparing Chou-Fasman parameters with analogous parameters determined from the results of conformational energy calculations of single amino acids. This comparison shows that Chou-Fasman parameters carry significant information about the environment of each amino acid. The success of the Chou-Fasman's prediction and the properties of the pair and triplet distribution of the amino acid residues suggest that every amino acid has a characteristic sequential residue environment in proteins. The observed preferences could be invoked, for example, in protein design or in the study of the evolutionary relationship of proteins.     

Comprehensive analysis of the numbers,lengths and amino acid compositions of transmembrane helices in prokaryotic,eukaryotic and viral integral membrane proteins of high-resolution structure

We report a comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in 235 high-resolution structures of integral membrane proteins. The properties of 1551 transmembrane helices in the structures were compared with those obtained by analysis of the same amino acid sequences using topology prediction tools. Explanations for the 81 (5.2%) missing or additional transmembrane helices in the prediction results were identified. Main reasons for missing transmembrane helices were mis-identification of N-terminal signal peptides, breaks in α-helix conformation or charged residues in the middle of transmembrane helices and transmembrane helices with unusual amino acid composition. The main reason for additional transmembrane helices was mis-identification of amphipathic helices, extramembrane helices or hairpin re-entrant loops. Transmembrane helix length had an overall median of 24 residues and an average of 24.9 ± 7.0 residues and the most common length was 23 residues. The overall content of residues in transmembrane helices as a percentage of the full proteins had a median of 56.8% and an average of 55.7 ± 16.0%. Amino acid composition was analysed for the full proteins, transmembrane helices and extramembrane regions. Individual proteins or types of proteins with transmembrane helices containing extremes in contents of individual amino acids or combinations of amino acids with similar physicochemical properties were identified and linked to structure and/or function. In addition to overall median and average values, all results were analysed for proteins originating from different types of organism (prokaryotic, eukaryotic, viral) and for subgroups of receptors, channels, transporters and others.     

QBES: predicting real values of solvent accessibility from sequences by efficient, constrained energy optimization

Solvent accessibility, one of the key properties of amino acid residues in proteins, can be used to assist protein structure prediction. Various approaches such as neural network, support vector machines, probability profiles, information theory, Bayesian theory, logistic function, and multiple linear regression have been developed for solvent accessibility prediction. In this article, a much simpler quadratic programming method based on the buriability parameter set of amino acid residues is developed. The new method, called QBES (Quadratic programming and Buriability Energy function for Solvent accessibility prediction), is reasonably accurate for predicting the real value of solvent accessibility. By using a dataset of 30 proteins to optimize three parameters, the average correlation coefficients between the predicted and actual solvent accessibility are about 0.5 for all four independent test sets ranging from 126 to 513 proteins. The method is efficient. It takes only 20 min for a regular PC to obtain results of 30 proteins with an average length of 263 amino acids. Although the proposed method is less accurate than a few more sophisticated methods based on neural network or support vector machines, this is the first attempt to predict solvent accessibility by energy optimization with constraints. Possible improvements and other applications of the method are discussed.     

Evaluating amber force fields using computed NMR chemical shifts

NMR chemical shifts can be computed from molecular dynamics (MD) simulations using a template matching approach and a library of conformers containing chemical shifts generated from ab initio quantum calculations. This approach has potential utility for evaluating the force fields that underlie these simulations. Imperfections in force fields generate flawed atomic coordinates. Chemical shifts obtained from flawed coordinates have errors that can be traced back to these imperfections. We use this approach to evaluate a series of AMBER force fields that have been refined over the course of two decades (ff94, ff96, ff99SB, ff14SB, ff14ipq, and ff15ipq). For each force field a series of MD simulations are carried out for eight model proteins. The calculated chemical shifts for the ¹H, ¹⁵N, and ¹³C^a atoms are compared with experimental values. Initial evaluations are based on root mean squared (RMS) errors at the protein level. These results are further refined based on secondary structure and the types of atoms involved in nonbonded interactions. The best chemical shift for identifying force field differences is the shift associated with peptide protons. Examination of the model proteins on a residue by residue basis reveals that force field performance is highly dependent on residue position. Examination of the time course of nonbonded interactions at these sites provides explanations for chemical shift differences at the atomic coordinate level. Results show that the newer ff14ipq and ff15ipq force fields developed with the implicitly polarized charge method perform better than the older force fields.     

Improved side‐chain torsion potentials for the Amber ff99SB protein force field

Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side‐chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha‐helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side‐chain torsion potentials of these residues to match new, high‐level quantum‐mechanical calculations. Finally, we used microsecond‐timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side‐chain conformations. The new force field, which we have termed Amber ff99SB‐ILDN, exhibits considerably better agreement with the NMR data. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Differentiation between two-state and multi-state folding proteins based on sequence

Prediction of protein-folding rates follows different rules in two-state and multi-state kinetics. The prerequisite for the prediction is to recognize the folding kinetic pathway of proteins. Here, we use the logistic regression and support vector machine to discriminate between two-state and multi-state folding proteins. We find that chain length is sufficient to accurately recognize multi-state proteins. There is a transition boundary between two kinetic models. Protein folds with multi-state kinetics, if its length is larger than 112 residues. The logistic prediction from amino acid composition shows that the kinetic pathway of folding is closely related to amino acid volume. Small amino acids make two-state folding easier, and vice versa. However, cysteine, alanine, arginine, lysine, histidine, and methionine do not conform to this rule.     

Effect of various kinds of dietary protein and supplementation with limiting amino acids on growth,haemolymph components and uric acid excretion in the silkworm, Bombyx mori

Effects of various kinds of dietary protein on growth of the silkworm, Bombyx mori, were determined using semi-synthetic diets. Also, the ingestion, digestion and utilization of dry matter and of nitrogen were measured. Nutritive effects of dietary proteins and supplementation of limiting amino acids on haemolymph protein and amino acids pattern were also investigated. Larval growth was largely dependent on the dietary proteins. When the larvae were reared on a diet containing weakly nutritive proteins such as gluten and zein, haemolymph protein was decreased and uric acid excretion was markedly accelerated. The free amino acid composition of the haemolymph manifested characteristic patterns according to the kinds of dietary protein.The supplementation of gluten and zein with their limiting amino acids resulted in a rise of haemolymph protein and a drop in uric acid excretion. The amino acid patterns in the haemolymph were greatly changed according to supplementation.     

氨基酸转运载体研究进展

氨基酸转运载体是介导氨基酸跨膜转运的膜蛋白,在氨基酸营养机体细胞和神经调节过程中起着重要作用;而且,其功能异常会导致严重的氨基酸吸收和代谢障碍性疾病,也具有重要的病理学意义。本文就近年来关于中性氨基酸、酸性氨基酸和碱性氨基酸转运载体家族成员及其组织分布、分子生物学特征、生理功能和病理学意义等研究进展进行了综述。     

Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils

The alpha-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of the coiled-coil motifs, including those modified by fluorination, several fluorinated and nonfluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER used a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, and ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly, better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled-coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine when compared with 5,5,5,5',5',5'-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5',5',5'-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may provide a driving force for coiled-coil dimer formation. Estimation of the ellipticity at 222 nm from a series of snapshots from the MD simulations with DicroCalc shows distinct increases in the ellipticity when the coiled-coil is fluorinated, which suggests that the helicity in the folded coiled-coils is greater when fluorinated.     

Exploring the environmental preference of weak interactions in (alpha/beta)8 barrel proteins

The environmental preference for the occurrence of noncanonical hydrogen bonding and cation-pi interactions, in a data set containing 71 nonredundant (alpha/beta)(8) barrel proteins, with respect to amino acid type, secondary structure, solvent accessibility, and stabilizing residues has been performed. Our analysis reveals some important findings, which include (a) higher contribution of weak interactions mediated by main-chain atoms irrespective of the amino acids involved; (b) domination of the aromatic amino acids among interactions involving side-chain atoms; (c) involvement of strands as the principal secondary structural unit, accommodating cross strand ion pair interaction and clustering of aromatic amino acid residues; (d) significant contribution to weak interactions occur in the solvent exposed areas of the protein; (e) majority of the interactions involve long-range contacts; (f) the preference of Arg is higher than Lys to form cation-pi interaction; and (g) probability of theoretically predicted stabilizing amino acid residues involved in weak interaction is higher for polar amino acids such as Trp, Glu, and Gln. On the whole, the present study reveals that the weak interactions contribute to the global stability of (alpha/beta)(8) TIM-barrel proteins in an environment-specific manner, which can possibly be exploited for protein engineering applications.     

N-Phosphoryl Amino Acids and Biomolecular Origins. Review Paper in Honor of the 50th Anniversary of the Publication of ``A Production of Amino Acids under Possible Primitive Earth Conditions' (Miller, 1953)

The possible role of phosphoryl amino acids for biomolecular origins is briefly reviewed. Peptide formation, ester formation, ester exchange on phosphorus and N to O migration occurred when the N-phosphoryl amino acid was incubated at room temperature. Short nucleotides and peptides were formed when nucleoside was reacted with N-phosphoryl amino acid at room temperature. Serine and threonine residues in their conjugate with different nucleosides (mediated with phosphorus) showed different self-cleavage activities. N-phosphoryl Histine and Ser-His dipeptide could cleave nucleic acids, proteins and esters in neutral medium. Based on a simple model, a pathway of 'co-evolution of protein and nucleic acid' was proposed.     

Generation of a cysteine sulfinic acid analog for incorporation in peptides using solid phase peptide synthesis

The sulfinic acid analog of aspartic acid, cysteine sulfinic acid, introduces a sulfur atom that perturbs the acidity and oxidation properties of aspartic acid. Cysteine sulfinic acids are often introduced in peptides and proteins by oxidation of cysteine, but this method is limited as all cysteine residues are oxidized and cysteine residues are often oxidized to sulfonic acids. To provide the foundation for the specific incorporation of cysteine sulfinic acids in peptides and proteins, we synthesized a 9-fluorenylmethyloxycarbonyl (Fmoc) benzothiazole sulfone analog. Oxidation conditions to generate the sulfone were examined and oxidation of the Fmoc-protected sulfide (3) with NbC in hydrogen peroxide provided the corresponding sulfone (4) in the highest yield and purity. Reduction with sodium borohydride generated the cysteine sulfinic acid (5) suggesting this approach may be an efficient method to incorporate a cysteine sulfinic acid in biomolecules. A model tripeptide bearing a cysteine sulfinic acid was synthesized using this approach. Future studies are aimed at using this method to incorporate cysteine sulfinic acids in peptide hormones and proteins for use in the study of biological function.     

Properties of integral membrane protein structures: derivation of an implicit membrane potential

Distributions of each amino acid in the trans-membrane domain were calculated as a function of the membrane normal using all currently available alpha-helical membrane protein structures with resolutions better than 4 A. The results were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans-membrane alpha-helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For alpha-helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans-membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials.     

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (b_KCl = 0.15 mol/kg) and high (b_KCl = 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.     

Water activity coefficients in aqueous amino acid solutions by molecular dynamics simulation: 1. Force field development

New force fields for molecular dynamics (MD) simulation of aqueous zwitterionic amino acid simulations were developed. These were especially designed to calculate activity coefficient of water in amino acid solutions with high accuracy. For example, aqueous solutions of the following amino acids were considered: glycine, alanine, α-aminobutyric acid, α-aminovalerianic acid, valine and leucine. The force fields were obtained by quantum chemical calculations using B3LYP/6-31G and MP2/6-311(d,p) model theories in combination with the Merz–Kollmann–Singh scheme. To further increase the accuracy of the force field, a polarised continuum was considered in all quantum chemical calculations. Water activity coefficients obtained from MD using different all-purpose literature force fields, namely, OPLS, AMBER ff03 and GROMOS 53A6 as well as experimental data are compared with the results utilising the new force field. The new force field is shown to give better results compared with experimental data than existing force fields.     

Prediction of protein domain boundaries from statistics of appearance of amino acid residues

A database of 452 two-domain proteins with less than 25% homology was constructed. One half of the database was used to obtain statistics on the appearance of amino acid residues at domain boundaries. Small and hydrophilic residues (proline, glycine, asparagine, glutamic acid, arginine, etc.) occurred more often at domain boundaries than in total proteins. Hydrophobic residues (tryptophan, methionine, phenylalanine, etc.) were rarer at domain boundaries than in total proteins. Probability scales of amino acid appearance in boundary-flanking regions were constructed with these statistics and used to predict the domain boundaries in proteins of the other half of the database. The probability scale obtained by averaging the appearance of amino acids over an 8-residue region (±4 residues from the real domain boundaries) yielded the best results: domain boundaries were predicted within 40 residues of the real boundary in 57% of proteins and within 20 residues of the real boundary in 41% of proteins. The probability scale was used to predict the domain boundaries in proteins with unknown structures (CASP6).     

Effect of co-evolving amino acid residues on topology of phylogenetic trees

The presence in proteins of amino acid residues that change in concert during evolution is associated with keeping constant the protein spatial structure and functions. As in the case with morphological features, correlated substitutions may become the cause of homoplasies--the independent evolution of identical non-homological adaptations. Our data obtained on model phylogenetic trees and corresponding sets of sequences have shown that the presence of correlated substitutions distorts the results of phylogenetic reconstructions. A method for accounting for co-evolving amino acid residues in phylogenetic analysis is proposed. According to this method, only a single site from the group of correlated amino acid positions should remain, whereas other positions should not be used in further phylogenetic analysis. Simulations performed have shown that replacement on the average of 8% of variable positions in a pair of model sequences by coordinately evolving amino acid residues is able to change the tree topology. The removal of such amino acid residues from sequences before phylogenetic analysis restores the correct topology.     

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

The rise of CuI‐catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in‐house preparation of a set of five Fmoc azido amino acids: β‐azido l ‐alanine and d ‐alanine, γ‐azido l ‐homoalanine, δ‐azido l ‐ornithine and ω‐azido l ‐lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user‐friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3 – 5 , 20 , 25 , 26 , 30 and 43 – 47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 μs. Four combinations of protein and water force fields were tested: ff19SB/OPC , ff19SB/TIP4P-D , ff03CMAP/TIP4P-D , and a99SB-disp/TIP4P-disp , with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 μs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and ³J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.