Properties Of Amino Acid Side Chains Research Articles

Quinolone Resistance in Gallibacterium anatis Determined by Mutations in Quinolone Resistance-Determining Region.

Control of the important pathogen, Gallibacterium anatis, which causes salpingitis and peritonitis in poultry, relies on treatment using antimicrobial compounds. Among these, quinolones and fluoroquinolones have been used extensively, leading to a rise in the prevalence of resistant strains. The molecular mechanisms leading to quinolone resistance, however, have not previously been described for G. anatis, which is the aim of this study. The present study combines phenotypic antimicrobial resistance data with genomic sequence data from a collection of G. anatis strains isolated from avian hosts between 1979 and 2020. Minimum inhibitory concentrations were determined for nalidixic acid, as well as for enrofloxacin for each included strain. In silico analyses included genome-wide queries for genes known to convey resistance towards quinolones, identification of variable positions in the primary structure of quinolone protein targets and structural prediction models. No resistance genes known to confer resistance to quinolones were identified. Yet, a total of nine positions in the quinolone target protein subunits (GyrA, GyrB, ParC and ParE) displayed substantial variation and were further analyzed. By combining variation patterns with observed resistance patterns, positions 83 and 87 in GyrA, as well as position 88 in ParC, appeared to be linked to increased resistance towards both quinolones included. As no notable differences in tertiary structure were observed between subunits of resistant and sensitive strains, the mechanism behind the observed resistance is likely due to subtle shifts in amino acid side chain properties.

Identification of Residues within Flavivirus Non-Structural Protein Associated with Mosquito Transmission

Flavivirus non-structural protein 1 (NS1) is present at high levels in patient blood and has been previously implicated in increasing virus acquisition by mosquito vectors, which may facilitate increased transmission. Therefore, flaviviruses transmitted by Aedes aegypti mosquitoes were hypothesised to have similarities within NS1, and potentially other NS proteins, that may correlate with mosquito-borne (rather than tick-borne or no vector) transmission. Representative flavivirus amino acid sequences were downloaded from the NCBI Protein database, aligned using viprbrc.org and then manually analysed to identify residues correlating with arthropod vector tropism. All available sequences for individual clinically important flaviviruses were then analysed on viprbrc.org to determine sequence conservation within viral species for those residues correlating with vector tropism. Overall, we found five residues in NS1 that were highly correlated with transmission by mosquitoes, had functionally relevant differences in amino acid side chain properties between mosquito- and tick-borne viruses and had conservation within viral species. Additionally, a further nine residues in NS3 and four residues in NS4A were identified using the same methodology. Importantly, for NS1, for which more data is available regarding its role in influencing transmission, the residues we identified have not been implicated in increased transmission thus far. Hence, future work would aim to test the impact of mutating these NS1 residues to elucidate whether they modulate NS1 secretion into the blood stream, virus replication and/or transmission. Gaining an understanding of the molecular determinants underpinning vector tropism could be useful in the creation of transmission-incompetent vectors.

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation.

Amino-acid side-chain properties in proteins are key determinants of protein function. NMR spin relaxation of side chains is an important source of information about local protein dynamics and flexibility. However, traditional solution NMR relaxation methods are most sensitive to sub-nanosecond dynamics lacking information on slower ns-μs time-scale motions. Nanoparticle-assisted NMR spin relaxation (NASR) of methyl-side chains is introduced here as a window into these ns-μs dynamics. NASR utilizes the transient and nonspecific interactions between folded proteins and slowly tumbling spherical nanoparticles (NPs), whereby the increase of the relaxation rates reflects motions on time scales from ps all the way to the overall tumbling correlation time of the NPs ranging from hundreds of ns to μs. The observed motional amplitude of each methyl group can then be expressed by a model-free NASR S2 order parameter. The method is demonstrated for 2H-relaxation of CH2D methyl moieties and cross-correlated relaxation of CH3 groups for proteins Im7 and ubiquitin in the presence of anionic silica-nanoparticles. Both types of relaxation experiments, dominated by either quadrupolar or dipolar interactions, yield highly consistent results. Im7 shows additional dynamics on the intermediate time scales taking place in a functionally important loop, whereas ubiquitin visits the majority of its conformational substates on the sub-ns time scale. These experimental observations are in good agreement with 4-10 μs all-atom molecular dynamics trajectories. NASR probes side-chain dynamics on a much wider range of motional time scales than previously possible, thereby providing new insights into the interplay between protein structure, dynamics, and molecular interactions that govern protein function.

Reflexivity, coding and quantum biology

Biological systems are fundamentally computational in that they process information in an apparently purposeful fashion rather than just transferring bits of it in a purely syntactical manner. Biological information, such has genetic information stored in DNA sequences, has semantic content. It carries meaning that is defined by the molecular context of its cellular environment. Information processing in biological systems displays an inherent reflexivity, a tendency for the computational information-processing to be “about” the behaviour of the molecules that participate in the computational process. This is most evident in the operation of the genetic code, where the specificity of the reactions catalysed by the aminoacyl-tRNA synthetase (aaRS) enzymes is required to be self-sustaining. A cell’s suite of aaRS enzymes completes a reflexively autocatalytic set of molecular components capable of making themselves through the operation of the code. This set requires the existence of a body of reflexive information to be stored in an organism’s genome. The genetic code is a reflexively self-organised mapping of the chemical properties of amino acid sidechains onto codon “tokens”. It is a highly evolved symbolic system of chemical self-description. Although molecular biological coding is generally portrayed in terms of classical bit-transfer events, various biochemical events explicitly require quantum coherence for their occurrence. Whether the implicit transfer of quantum information, qbits, is indicative of wide-ranging quantum computation in living systems is currently the subject of extensive investigation and speculation in the field of Quantum Biology.

Novel 3D bio-macromolecular bilinear descriptors for protein science: Predicting protein structural classes.

In the present study, we introduce novel 3D protein descriptors based on the bilinear algebraic form in the ℝ(n) space on the coulombic matrix. For the calculation of these descriptors, macromolecular vectors belonging to ℝ(n) space, whose components represent certain amino acid side-chain properties, were used as weighting schemes. Generalization approaches for the calculation of inter-amino acidic residue spatial distances based on Minkowski metrics are proposed. The simple- and double-stochastic schemes were defined as approaches to normalize the coulombic matrix. The local-fragment indices for both amino acid-types and amino acid-groups are presented in order to permit characterizing fragments of interest in proteins. On the other hand, with the objective of taking into account specific interactions among amino acids in global or local indices, geometric and topological cut-offs are defined. To assess the utility of global and local indices a classification model for the prediction of the major four protein structural classes, was built with the Linear Discriminant Analysis (LDA) technique. The developed LDA-model correctly classifies the 92.6% and 92.7% of the proteins on the training and test sets, respectively. The obtained model showed high values of the generalized square correlation coefficient (GC(2)) on both the training and test series. The statistical parameters derived from the internal and external validation procedures demonstrate the robustness, stability and the high predictive power of the proposed model. The performance of the LDA-model demonstrates the capability of the proposed indices not only to codify relevant biochemical information related to the structural classes of proteins, but also to yield suitable interpretability. It is anticipated that the current method will benefit the prediction of other protein attributes or functions.

Intrinsic energy landscapes of amino acid side-chains.

Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.

Conformational distributions of denatured and unstructured proteins are similar to those of 20 × 20 blocked dipeptides

Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out (1)H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH(2)), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.

Sulfonylurea Receptor Transmembrane Domain Zero Mutations that Disrupt Full Length and Minimal ATP-Sensitive Potassium Channel Properties

Kir6.2 subunits constitute the pore-domain of the ATP-sensitive potassium channel (KATP) and, as such, are acted upon by accessory sulfonylurea receptor (SUR1) subunits to transduce ligand and pharmacologic signals into channel activity modifications. It is the interface of these two subunits that this work attempts to illuminate. We recently characterized two disease-causing mutations positioned in the first transmembrane domain of SUR1 (R74W and E128K located in TMD0) that decrease both ATP-sensitive inhibition and intrinsic open-probability (Po) of KATP. Because TMD0 has been shown to endow KATP channels with increased Po, we hypothesized that R74 and E128 lie at the subunit-subunit interface between SUR1 and Kir6.2 and their mutation leads to decreased SUR1-Kir6.2 interactions. We first characterized the amino-acid side-chain properties of R74x and E128x that determine channel surface expression and ATP-sensitive inhibition via a mutagenesis-based screen. Aromatic residues at R74 resulted in dramatic reduction of ATP-induced inhibition (IC50) whereas any non-charge conserving residue caused significant loss of surface expression. E128x mutations that decreased the ATP IC50 caused a parallel reduction of surface expression; residue charge, hydrophobicity, or size were independent of this relationship. To more directly assess TMD0SUR1-Kir6.2 stability, we compared single channel voltage-clamp recordings of channels formed by Kir6.2 alone (Kir6.2delta35C ) to minimal KATP channels (i.e., TMD0 + Kir6.2delta35C) with and without R74W or E128K mutations. Intrinsic open probabilities of mutant minimal channels were significantly less than WT TMD0+Kir6.2delta35C, yet not less than Kir6.2deltaC alone. Our results support the hypothesis that contacts between TMD0 and Kir6.2_rather than the influence of SUR1 regions terminal to TMD0_are disrupted by introduction of mutations at R74 and E128.

Nearest‐neighbor effects and structural preferences in dipeptides are a function of the electronic properties of amino acid side‐chains

The electronic properties of amino acid side-chains are emerging as an important factor in the preference for secondary structure in proteins. These properties have not been fully characterized, nor has their role in the behavior of peptides been explored in any detail. The present studies sought to evaluate several possibilities: 1) that hydrophilicity can be expressed solely in electronic terms, 2) that substituent effects of side-chains extend across the peptide bond, and (3) nearest-neighbor effects in dipeptides correlate with secondary structural preferences. Quantum mechanics (QM) calculations were used to define the electronic properties of individual amino acids and dipeptides. It was found that the hydrophilicity of an amino acid side-chain can be accurately represented as a function of the electron densities of its component atoms. In addition, the nature of an amino acid in the second position of a dipeptide affects the electronic properties (Mulliken populations and electron densities) of the main-chain atoms of the first residue. Certain electronic features of the dipeptides strongly correlated with propensity for secondary structure. Specifically, Mulliken population data at the Calpha atom and N atom predicted preference for alpha-helices versus coil and strand conformations, respectively. Analysis of dipeptides arrayed in either helical or extended structures revealed lengthening of main-chain bonds in the alpha-helical conformations. A thorough characterization of the electronic properties of amino acids and short peptide segments may provide a better understanding of the forces that determine secondary structure in proteins.

Adenine recognition: a motif present in ATP-, CoA-, NAD-, NADP-, and FAD-dependent proteins.

Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA.

Blockage of Human T Lymphocyte Kv1.3 Channels by Pi1, a Novel Class of Scorpion Toxin

Using the patch-clamp technique we determined that Pandinus imperator toxin Pi1, a recently described peptide toxin having four disulfide bridges instead of the usual three in scorpion toxins, blocked Kv1.3 channels of human T lymphocytes from the extracellular side with a 1:1 stoichiometry. Kv1.3 block was instantaneous and removable with toxin-free extracellular solution. The toxin did not influence activation or inactivation of the channels. We found that Pi1 blocked Kv1.3 with less affinity (Kd = 11.4 nM) than the structurally related three disulfide bridge containing toxins Pi2 (50 pM) and Pi3 (0.5 nM). The fourth disulfide bridge in Pi1 had no influence on the channel binding ability of the toxin; the less effective block was due to differences in amino acid side chain properties at positions 11 and 35.

Theoretical indicators of enzyme reaction specificity from conserved information in amino acid sidechains.

Amino acid sequences for 11 acetohydroxy acid synthase (EC 4.1.3.18; AHS) polypeptides with experimentally established activity were chosen for computational comparisons to detect conserved local information associated with reaction specificity for each sequence. Windowed analysis by Pearson product moment cross-correlation of six amino acid sidechain properties revealed locally conserved segments common to all proteins with AHS activity. Seven information segments were detected in the same arrangement in sequences for the large subunit polypeptides of prokaryotes, and in the sequences for single polypeptides of eukaryotic AHS. The information segments were numbered 1-7 according to sequential position, and sequence features such as cofactor binding sites were defined for specific segments. Extension of the information segment analysis to seven other proteins of the pyruvate decarboxylase superfamily permitted use of the content and organization of information segments to recognize four classes of enzyme reaction specificity. Estimates of information entropy, based upon a state space defined by reaction specificity, directly reflected the known reaction complexity for all but one enzyme examined. Our data suggest that development of information-segment models for enzyme superfamilies may improve the accuracy of inferring protein activity from sequence.

Conformation and Antimicrobial Activity of Linear Derivatives of Tachyplesin Lacking Disulfide Bonds

Tachyplesin is a potent antimicrobial peptide isolated from the hemocytes of the horseshoe crab,Tachypleus tridentatus.Previous studies have shown that the 17-residue peptide has an intrinsic amphipathic structure conferred by two antiparallel β-sheets held rigidly by two disulfide bonds. Taking its short length into account and the potential of such a small polypeptide to take on multiple conformational states, one may assume that the disulfide bonds are relevant determinants of function. However, in order to gain a global perspective on the tolerance of cysteine residues in tachyplesin to amino acid substitutions, a series of linear peptides have been synthesized and their physicochemical properties analyzed. In these linear peptides, the cysteines have been replaced with amino acids possessing different side-chain properties, i.e., aliphatic hydrophobic (Ala, Leu, Ile, Val, and Met), aromatic hydrophobic (Phe and Tyr), and acidic (Asp). Activity assays using natural and synthetic membranes, and conformational measurements, highlight the subtle influence and variability of the amino acid side-chain properties on peptide structure. While an unequivocal interpretation of the results will have to await more refined structural measurements, our results indicate that a rigidly held disulfide-bonded β-pleated sheet structure may not be absolutely essential for antimicrobial activity. Furthermore, the results challenge the accepted dogma of structure–activity relationships among antimicrobial peptides and suggest that the maintenance of peptide hydrophobic–hydrophilic balance may be a critical parameter, in addition to structure, in the design of peptides with pharmaceutical relevance.

Amino acid substitutions in structurally related proteins a pattern recognition approach: Determination of a new and efficient scoring matrix

Amino acid substitutions in evolutionarily related proteins have been studied from a structural point of view. We consider here that an amino acid al in a protein p1 has been replaced by the amino acid a2 in the structurally similar protein p2 if, after superposition of the p1 and p2 structures, the al and a2 C α atoms are no more than 1.2 Å apart. Thirty-two proteins, grouped in 11 classes, have been analysed by this method. This produced 2860 amino acid pairs (substitutions), which were analysed by multi-dimensional statistical methods. The main results are as follows: (1) according to the observed exchangeability of amino acid side-chains, only four groups (strong clusters) could be delineated; (i) Ile and Val, (ii) Leu and Met, (iii) Lys, Arg and Gln, and (iv) Tyr and Phe. The other residues could not be classified. (2) The matrix of distances between amino acids, or scoring matrix, determined from this study, is different from any other published matrix. (3) Except for the distance matrices based on the chemical properties of amino acid side-chains, which can be grouped together, all other published matrices are different from one another. (4) The distance matrix determined in this study seems to be very efficient for aligning distantly related protein sequences.

Comparison of scales of amino acid side chain properties by conservation during evolution of four proteins.

As the amino acid sequence of a given protein changes along the phylogenetic tree, enough of the overall folding pattern must be conserved to ensure that the protein still fulfils its biological function. Eighteen published scales which tabulate various side chain properties are compared here by computing the variance of each scale when applied to each of several protein families. The conservation of each scale of side chain properties is examined for the 20,627 residues in 60 mammalian myoglobins, 31 mammalian ribonucleases, insulin A and B chains (29 sequences each), 29 vertebrate and 28 plant cytochrome c's. Those scales which are the most highly conserved through the evolution of each protein family may well be the best predictors of protein folding patterns. The mean-area-buried scale and the optimized matching hydrophobicities scale are more conserved than other scales. An additional result is the relatively poor conservation across evolution of the Chou-Fasman secondary structure predictors.