Charge Of Amino Acid Residue Research Articles

Structural and Functional Characterization of β−lytic Protease from Lysobacter capsici VKM B−2533T

The crystal structure of the Lysobacter capsici VKM B-2533T β-lytic protease (Blp), a medicinally promising antimicrobial enzyme, was first solved. Blp was established to possess a folding characteristic of the M23 protease family. The groove of the Blp active site, as compared with that of the LasA structural homologue from Pseudomonas aeruginosa, was found to have amino acid differences. Biochemical analysis revealed no differences in the optimal reaction conditions for manifesting Blp and LasA bacteriolytic activities. At the same time, Blp had a broader range of action against living and autoclaved target cells. The results suggest that the distinction in the geometry of the active site and the charge of amino acid residues that form the active site groove can be important for the hydrolysis of different peptidoglycan types in target cells.

Calculation of Na+/Cl− Selectivity in Tight Junctions between Epithelial Cells by Methods of Nonlocal Electrostatics with Pole Models of Dielectric Function without an Overscreening Effect

A mathematical model of Na+/Cl− selectivity in tight junctions (TJs) between epithelial cells was developed. It was demonstrated that Na+/Cl− selectivity in TJs depends on the total charge of amino-acid residues of claudin macromolecules within TJs, as well as on the ion-distribution coefficients between TJs and free solution. It was demonstrated that the obtained formulas predict a change of Na+/Cl− selectivity in TJs for Cl−/Na+ selectivity if the sign of the total charge of amino-acid residues in TJ changes. The calculated Na+/Cl− selectivity value for MDCK cells coincides with the experimental data of (Colegio et al., 2002). To calculate a change in ion-solvation energy during their transition to TJs, formulas for nonlocal electrostatics with one-, two-, and three-pole models of dielectric function without taking into account the overscreening effect were used.

Gene duplications and the evolution of c-type lysozyme during adaptive radiation of East African cichlid fish

The adaptive radiation of African cichlids is a prime model system for studying vertebrate speciation. Cichlid species are distributed in lakes and rivers with various water conditions such as various pH values. The innate immune system in fish is particularly important because water contains a wide range of pathogenic microorganisms. To investigate the evolution of the host defense system in cichlids, we isolated the c-type lysozyme gene, which functions in the innate immune system of fish. Southern blot and sequence analyses showed that the lysozyme gene underwent several gene duplication events and evolved with amino acid replacements during the adaptive radiation of cichlids. The inferred 3D structure revealed that the amino acid substitutions were localized on the lysozyme surface. Moreover, more than half of the surface substitutions changed the charge of amino acid residues, suggesting changes in the optimum pH for enzymatic activity. In African cichlids, the lysozyme genes may have played and still play an important role in defense against pathogens.

Quantum Mechanics/Molecular Mechanics Restrained Electrostatic Potential Fitting

We present a quantum mechanics/molecular mechanics (QM/MM) method to evaluate the partial charges of amino acid residues for use in MM potentials based on their protein environment. For each residue of interest, the nearby residues are included in the QM system while the rest of the protein is treated at the MM level of theory. After a short structural optimization, the partial charges of the central residue are fit to the electrostatic potential using the restrained electrostatic potential (RESP) method. The resulting charges and electrostatic potential account for the individual environment of the residue, although they lack the transferable nature of library partial charges. To evaluate the quality of the QM/MM RESP charges, thermodynamic integration is used to measure the pKa shift of the aspartic acid residues in three different proteins, turkey egg lysozyme, beta-cryptogein, and Thioredoxin. Compared to the AMBER ff99SB library values, the QM/MM RESP charges show better agreement between the calculated and experimental pK(a) values for almost all of the residues considered.

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.

Creation of Interferon-α8 Mutants with Amino Acid Substitutions Against Interferon-α Receptor-2 Binding Sites Using Phage Display System and Evaluation of Their Biologic Properties

In this study, we describe the creation of three interferon-alpha (IFN-alpha)8 mutants with markedly higher antiviral and antiproliferative activities in comparison with those of the wild-type (wt)IFN-alpha8, wtIFN-alpha2, and IFN-con1 using a phage display system. Sequence analysis showed that three out of the six hot-spot amino acid residues of wtIFN-alpha8 known to be important for the interaction with the IFN-alpha receptor-2 (IFNAR-2)-binding sites were substituted to other amino acids and the others remained. Although affinity analysis revealed that the dissociation constant (K(D)) of IFN-alpha8 mutants was almost the same with that of wtIFN-alpha8, furthermore, the rates of association (k(a)) and dissociation (k(d)) were relatively lower. These results suggest that changes in the surface electronic charge of amino acid residues lead to changes in binding affinity and kinetics (prolonged dissociation time) toward the IFNAR-2, resulting in the modification of the biological activity. Moreover, our results demonstrate that the molecular engineering of the IFN-alpha8 provides important insight into action of IFN and also it would be useful in the development of therapeutically prominent IFN preparations than those used in clinical practice.

Origin and evolution of viral interleukin-10 and other DNA virus genes with vertebrate homologues.

Phylogenies of gene families including members in both vertebrates and DNA viruses of the poxvirus and/or herpesvirus families showed that the viral genes originated at widely different times over the history of life. Certain of these viral genes (for example, the genes encoding the large and small subunits of ribonucleoside-diphosphate reductase) originated before animals diverged from fungi, while others originated much more recently. The most striking examples of recent origin involved viral genes encoding the cytokine interleukin-10 (IL-10), which originated independently in viruses at least three times since the divergence of the orders of eutherian mammals, presumably by viral capture of host genes. In certain domains, viral IL-10 genes showed significantly higher rates of nonsynonymous substitution than their nearest mammalian homologues. Though the mutation rate in these viral genes is up to 20 times that of the corresponding mammalian genes, a high mutation rate alone did not account for these differences because they were not seen in all domains. Rather, in certain domains it appears that functional constraints present in the case of mammalian IL-10 are relaxed in the case of the viral homologues. Furthermore, a nonrandom pattern of change with respect to amino acid residue charge in the N-terminal portion of the mature protein has occurred repeatedly in independently derived viral IL-10 genes, strongly suggesting that positive selection has led to divergence of this functionally important domain in viral IL-10.

Evolution of Cysteine Proteinases in Eukaryotes

Phylogenetic analyses of the major family of cysteine proteinases of eukaryotes revealed that this family has been characterized by numerous gene duplications, several of which occurred very early in eukaryote evolutionary history. For example, certain cysteine proteinases from plants and animals are homologous to each of the two cysteine proteinases of the protist Dictyostelium discoideum, indicating that these genes duplicated prior to the divergence of Dictyostelium, plants, and animals. All members of this gene family from metazoan parasites clustered in a well-defined subfamily with mammalian cathepsin B, whereas all those from protist parasites clustered outside this subfamily. Five members of this subfamily from Haemonchus contortus (a nematode parasite of sheep and other ruminants) evolved by gene duplications which were estimated to have occurred within the past 200 My. The proteins encoded by these five genes show a surprising number of residue charge differences from each other, particularly in helical regions in or near the substrate-binding cleft. Statistical analysis of DNA sequences showed that nonsynonymous nucleotide differences that cause amino acid residue charge changes have occurred in these regions at a greater rate than expected under random substitution, suggesting that these genes have diversified as a result of positive natural selection favoring such changes.

Coevolution of the vertebrate integrin alpha- and beta-chain genes.

The integrin receptors are heterodimers whose alpha and beta subunits are encoded by separate, evolutionarily unrelated multigene families. Phylogenetic analysis of DNA sequences from these two gene families showed that they have not always evolved in a parallel fashion. The integrin alpha chains that can form heterodimers with beta 1 do not constitute a monophyletic group, nor do the beta chains which can form heterodimers with alpha V. On the other hand, the vertebrate alpha chains associating with beta 2 are a monophyletic group. In the metal cation-binding region of the alpha chain, an exon exchange took place between human alpha M and alpha X approximately 40-50 Mya, homogenizing this functionally important region in these two alpha chains. When integrin beta chains of different functional classes are compared, nonsynonymous (amino acid altering) nucleotide substitutions that alter amino acid residue charge in the central region of the molecule occur at a rate significantly higher than that expected under random replacement. By contrast, when closely related beta 1 chains are compared, residue charge is conserved in this region. These results pinpoint the central region as a focus of functional divergence among integrin beta chains, perhaps relating to the ability of each beta integrin class to associate with a specific array of alpha integrins. Furthermore, they imply that positive, directional selection on this region has occurred in the evolution of the integrin beta-chain gene family.

Toward accurate transferable electrostatic models for polypeptides: A distributed multipole study of blocked amino acid residue charge distributions

AbstractWe have developed a method for building up accurate electrostatic models for polypeptides, based on a distributed multipole representation of the SCF charge densities for the dipeptides (CH3.CO.NH.CHR.CO.NH.CH3) of the naturally occurring amino acids. It is based on the observation that each peptide residue has almost the correct formal charge (0, ±1). We find that the variations in the backbone charge distributions (excluding proline) with sidechain have a negligible effect on the predicted electrostatic potential around the residue. However changes in the atomic multipoles with the torsion angles (ϕ, ψ, χ) are more significant, and may need to be taken into account if electrostatic potential close to the residue is required to high accuracy. This type of DMA peptide library provides more accurate, more theoretically based, estimates of the electrostatic potential around polypeptides than current models.

Circumsporozoite protein genes of malaria parasites (Plasmodium spp.): evidence for positive selection on immunogenic regions.

The circumsporozoite (CS) protein is a cell surface protein of the sporozoite, the stage of the life cycle of malaria parasites (Plasmodium spp.) that infects the vertebrate host. Analysis of DNA sequences supports the hypothesis that in Plasmodium falciparum, positive Darwinian selection favors diversity in the T-cell epitopes (peptides presented to T cells by host MHC molecules) of the CS protein. In gene regions encoding T cell epitopes of P. falciparum, the rate of nonsynonymous nucleotide substitution is significantly higher than that of synonymous substitution, whereas this is not true of other gene regions. Furthermore nonsynonymous nucleotide substitutions in these regions cause a change of amino acid residue charge significantly more frequently than expected by chance. By contrast, in Plasmodium cynomolgi, the same regions show no evidence of positive selection, and residue charge is conserved. The CS protein has a central repeat region, which is the target of host antibodies. In P. falciparum, the amino acid sequence of the repeat region is conserved within and between alleles. In P. cynomolgi, on the other hand, there is evidence that positive selection has favored evolution of two different repeat types within a given allele.