Specificity and mode of action of the muscle-type protein-arginine deiminase.

The primary and secondary specificities and mode of action of the muscle-type protein-arginine deiminase (PAD) were investigated using various derivatives of Arg and its homologues, as well as Arg-containing peptides by quantitative analyses of the reaction products on reverse-phase HPLC. The enzyme converted benzoyl-D-Arg-p-nitroanilide into its citrulline derivative at 18% of the rate of the L-isomer, while the D-Arg residues in peptides were not deiminated to a significant extent. This suggests that PAD does not have strict stereospecificity and it is dependent on the structure of the residues or groups on both sides of the target Arg residue. In contrast, the benzoyl-/-ethyl ester derivatives of homoarginine, alpha-amino-beta-guanidino-propionic acid, canavanine, and NG-methyl-Arg, exhibited poor PAD susceptibility, suggesting that the length and nature of the arm as exactly three CH2 groups, and the integrity of the guanidyl group are quite strict specificity determinants. The enzyme action on Arg residues in peptides depends greatly on their position in the sequence, and on the nature of the neighboring residues. For example, deimination of Arg residues situated at positions 1-3 from the NH2-terminus, except for those preceded by a carbobenzoxy- or benzoyl-group, were in most cases very slow, whereas those at the COOH-terminus were deiminated relatively faster. A single Arg residue sandwiched between two Pro residues was not deiminated at all, while a pair of Arg residues between two Pro were deiminated moderately. Consequently, PAD exhibited a variety of modes of action on more than one Arg residues in the peptides tested. The results suggest the applicability of PAD, albeit quite limited, for selective modification of certain Arg residues in peptides and proteins by appropriately controlling reaction time and several other parameters. The PAD's mode of action was compared with those of three Arg-bond cleaving proteases.     

Look-up tables for protein solvent accessibility prediction and nearest neighbor effect analysis

We developed dictionaries of two-, three-, and five-residue patterns in proteins and computed the average solvent accessibility of the central residues in their native proteins. These dictionaries serve as a look-up table for making subsequent predictions of solvent accessibility of amino acid residues. We find that predictions made in this way are very close to those made using more sophisticated methods of solvent accessibility prediction. We also analyzed the effect of immediate neighbors on the solvent accessibility of residues. This helps us in understanding how the same residue type may have different accessible surface areas in different proteins and in different positions of the same protein. We observe that certain residues have a tendency to increase or decrease the solvent accessibility of their neighboring residues in C- or N-terminal positions. Interestingly, the C-terminal and N-terminal neighbor residues are found to have asymmetric roles in modifying solvent accessibility of residues. As expected, similar neighbors enhance the hydrophobic or hydrophilic character of residues. Detailed look-up tables are provided on the web at www.netasa.org/look-up/.     

Contributions of the ionization states of acidic residues to the stability of the coiled coil domain of matrilin-1

The pKa values of eight glutamic acid residues in the homotrimeric coiled coil domain of chicken matrilin-1 have been determined from 2D H(CA)CO NMR spectra recorded as a function of the solution pH. The pKa values span a range between 4.0 and 4.7, close to or above those for glutamic acid residues in unstructured polypeptides. These results suggest only small favorable contributions to the stability of the coiled coil from the ionization of its acidic residues.     

Plasma-Membrane lipid composition and ethanol tolerance inSaccharomyces cerevisiae

Populations of cells suspended anaerobically in buffered (pH 4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration.     

Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions.     

Protein-DNA interactions: structural, thermodynamic and clustering patterns of conserved residues in DNA-binding proteins

Amino acid residues, which play important roles in protein function, are often conserved. Here, we analyze thermodynamic and structural data of protein-DNA interactions to explore a relationship between free energy, sequence conservation and structural cooperativity. We observe that the most stabilizing residues or putative hotspots are those which occur as clusters of conserved residues. The higher packing density of the clusters and available experimental thermodynamic data of mutations suggest cooperativity between conserved residues in the clusters. Conserved singlets contribute to the stability of protein-DNA complexes to a lesser extent. We also analyze structural features of conserved residues and their clusters and examine their role in identifying DNA-binding sites. We show that about half of the observed conserved residue clusters are in the interface with the DNA, which could be identified from their amino acid composition; whereas the remaining clusters are at the protein-protein or protein-ligand interface, or embedded in the structural scaffolds. In protein-protein interfaces, conserved residues are highly correlated with experimental residue hotspots, contributing dominantly and often cooperatively to the stability of protein-protein complexes. Overall, the conservation patterns of the stabilizing residues in DNA-binding proteins also highlight the significance of clustering as compared to single residue conservation.     

Crystal structure of tobacco necrosis virus at 2.25 A resolution

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Probing dimer interface stabilization within a four-helix bundle of the GrpE protein from Escherichia coli via internal deletion mutants: conversion of a dimer to monomer

Insight into protein stability and folding remains an important area for protein research, in particular protein-protein interactions and the self-assembly of homodimers. The GrpE protein from Escherichia coli is a homodimer with a four-helix bundle at the dimer interface. Each monomer contributes a helix-loop-helix to the bundle. To probe the interface stabilization requirements, in terms of the amount of buried residues in the bundle necessary for dimer formation, internal deletion mutants (IDMs) were created that sequentially truncate each of the two helices in the helix-loop-helix region. Circular dichroism (CD) spectroscopy showed that all IDM's still contained a significant amount of α-helical secondary structure. IDM's that contained 11 or fewer of 22 residues originally present in the helices, or those that lost at least 50% of residues with less than 20% the solvent accessible surfaces (that is, hydrophobic residues) were unable to form a significant amount of dimer species as shown by chemical cross-linking. Gel filtration studies of IDM3.0 (one that retains 10 residues in each helix) show this variant to be mainly monomeric.     

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     

Analysis of antibodies of known structure suggests a lack of correspondence between the residues in contact with the antigen and those modified by somatic hypermutation

Forty unique murine antibody-antigen complexes determined at 2.5 A or less resolution are analyzed to determine whether the residues in direct contact with the antigen are modified by somatic hypermutation. This was done by taking advantage of the recent characterization of the pool of Vkappa germline genes of the mouse. The average number of residues in contact with the antigen in the V(L) gene, which contains the CDRL-1, CDRL-2, and all but one residue of CDRL-3, was six. The average number of somatic mutations was similar (around five). However, as many as 53% of the antibodies did not show somatic replacements of residues in contact with the antigen. Another 28% had only one. Overall, the frequency of antibodies with increasing number of somatic replacements in residues in contact with the antigen decreased exponentially. A possible explanation of this finding is that mutations in the contacting residues have an adverse effect on the antigen-antibody interaction. This implies that most of the observed mutations are those remaining after negative (purifying) selection. Therefore, efficient strategies of site-directed mutagenesis to improve the affinity of antibodies should be focused on residues other than those directly interacting with the antigen.     

Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.     

Carboxylate groups in bovine somatotropin involved in growth promoting activity. Theoretical models based upon individual kinetic constants to interpret the activity decay after chemical modification

Modification of approximately one fifth of the carboxylate groups in bovine somatotropin with a water soluble carbodiimide caused loss of growth promoting potency pointing to the existence of residues related to the hormonal activity among those belonging to a fast reacting set. A sigmoidal curve was obtained whether the inactivation process was referred to reaction time or degree of modification. Isoelectrofocusing of derivatives released the native hormone from responsibility for the biological potency exerted by preparations with 1.5-2.6 modified carboxylate groups. Examination of the individual reaction kinetics of the 11 fast reacting residues, in turn, excluded the possibility of the sigmoidal character of the inactivation curve being caused by a nonexponential disappearance of essential residues, as a possible consequence of the chemical modification of others. According to synthetic models, the experimental curve may be the consequence of the effect of cumulative modification of 2 or 3 out of a set of 3 to 8 relevant residues.     

Biosynthesis of yeast glycoproteins. Processing of the oligosaccharides transferred from dolichol derivatives.

The oligosaccharides previously bound to dolichol diphosphate were isolated from Saccharomyces cerevisiae cells incubated with [U-14C]glucose. Five compounds were obtained that migrated with RGlucose of 0.100, 0.120, 0.145, 0.180, and 0.215 on paper chromatography. All of them contained mannose and 2 N-acetylhexosamine residues. The substances that migrated with the three lower RGlucose values had, in addition, glucose units. The structure of the oligosacchardies was very similar if not identical with that of the oligosaccharides isolated from the dolichol diphosphate derivatives synthesized "in vitro" by yeast or rat liver particulate preparations or "in vivo" by dog thyroid or rat liver slices as judged by their migration on paper chromatography, monosaccharide composition, and degradation compounds produced by alpha-mannosidase treatment or acetolysis. The oligosaccharides previously bound to asparagine residues in proteins were isolated from yeast cells which had been pulsed with [U-14C]glucose and chased with medium containing the unlabeled monosaccharide. The samples taken after very short pulses contained four oligosaccharides that migrated with RGlucose of 0.100, 0.120, 0.145, and 0.180 on paper chromatography. The first three compounds contained glucose, mannose, and 2 N-acetylhexosamine residues whereas the one that migrated with a RGlucose of 0.180 was devoid of the former monosaccharide. Samples taken after short chase periods revealed that the compounds that migrated with the lower RGlucose values gradually disappeared and were converted to the oligosaccharide with the higher RGlucose value was they lost their glucose residues. Similar analysis as those mentioned above showed that the structures of these compounds were similar to those of the dolichol diphosphate-bound oligosaccharides. Samples taken after longer chase periods revealed that the oligosaccharide that migrated with a RGlucose of 0.180 was subsequently either enlarged by the addition of more mannose residues or trimmed to smaller sizes.     

Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.     

农药的轭合与结合态残留

随着科技水平的不断提高,人们对农药残留影响生态环境的认识不断在更新和完善。农药的轭合与结合残留是在标记农药的出现和高精度分析仪器和技术得到应用后被发现和认识的。国外在这方面的研究开展得较早,工作也较     

Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: effects of the solvent dielectric.

We have investigated the spontaneous degradation of aspartate and asparagine residues via succinimide intermediates in model peptides in organic co-solvents. We find that the rate of deamidation at asparagine residues is markedly reduced in solvents of low dielectric strength. Theoretical considerations suggest that this decrease in rate is due to the destabilization of the deprotonated peptide bond nitrogen anion that is the postulated attacking species in succinimide formation. This result suggests that asparagine residues in regions with low dielectric constants, such as the interior of a protein or in a membrane bilayer, are protected from this type of degradation reaction. On the other hand, we found little or no effect on the rate of succinimide-mediated isomerization of aspartate residues when subjected to the same changes in dielectric constant. In this case, the destabilization of the attacking peptide bond nitrogen anion may be balanced by increased protonation of the aspartyl side chain carboxyl group, a reaction that results in a superior leaving group. Consequently, any protein structure or conformation that would increase the protonation of an aspartate side chain carboxyl group can be expected to render that residue more labile. These results may help explain why particular aspartate residues have been found to degrade in proteins at rates comparable to those of asparagine residues, even though aspartyl-containing peptides degrade more slowly than corresponding asparaginyl-containing peptides in aqueous solutions.     

Mutational analysis of autonomously functioning trans-activating peptides encoded by adenovirus E1A: evidence for two subdomains.

We have shown previously that a chemically synthesized adenovirus E1A region 3 peptide of 49 amino acids, protein domain 3 (PD3; residues 140 to 188 of the 289-amino-acid protein), trans activates viral genes in vitro and in vivo. To study structure-function relationships, we synthesized N-terminal deletion and cysteine substitution mutant peptides and tested their activities in a cell microinjection assay. Peptides lacking 1 to 12 N-terminal residues exhibited 5- to 50-fold-reduced molar specific activities, whereas those lacking 16 or 18 residues were inactive. Substitution of each of five PD3 cysteine residues with alanine resulted in substantial losses of activity: mutants in the PD3 N-terminal portion showed 40 to 55% of wild-type activity but required a 20-fold-higher concentration than PD3, whereas those in the C-terminal half were as much less active. These peptide mutant studies suggest the existence of two PD3 functional regions: one, localized in the C-terminal 70 to 75% of the molecule, is essential for trans activation; the other, localized in the N-terminal 25 to 30%, can be overridden to a significant extent at high peptide concentrations.