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At least 19 recordsLinked to original sources
PubMed1994

Stabilization and enhanced enzymatic activities of a mutant human lysozyme C77/95A with a cavity space by amino acid substitution.

Amino acid substitutions were examined to increase the stability of the mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by the formation of a hydrogen bond, five amino acid-substituted mutants, C77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and constructed based on computer graphics investigations for stabilizing the mutant protein. The values of the melting temperatures, Tm, at pH 3.0 of the two mutant proteins C77LC95A and C77/95S were increased by 2.9 degrees C and 2.3 degrees C, respectively, as compared to that of C77/95A. The C77IC95A and C77AC95L proteins showed almost the same stability as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 and 95 with the side residue of Leu77 in C77LC95A, and by the formation of a hydrogen bond between Ser77 and Ser95 in C77/95S. On the other hand, the substitution with isoleucine at 95 (C77AC95I) decreased the stability. The activities of the five mutant proteins against the synthetic substrate, p-nitrophenyl tetra-N-acetyl-beta-chitopentaoside, were higher than that of the wild-type human lysozyme, while the lytic activities against M. lysodeikticus were decreased in C77LC95A and C77IC95A, and increased in C77AC95L.

Base Sequence↗
PubMed1992

[Uneven distribution of amino acid substitutions throughout the amino acid sequence of homologous proteins].

A set of aligned homologous protein sequences is divided into two groups consisting of m and n most related sequences. The value of position variability for homologous protein sequences is defined as a number of failures to coincide in the intergroup comparison of all possible m*n pairs of amino acid residues in that position divided by m*n. The position variability value plotted versus the sequence position number with a window of 10 positions gives the intergroup local variability profile. Area S of the figure included between the local variability profile and the straight line corresponding to the mean local variability value is compared with the average area Sr for 1000 random homologous protein families. If S is greater than Sr by more than 2 standard deviation units sigma r, the local variability profile is assumed to contain peaks and hollows corresponding to significant variable and conservative regions of the sequences. The profile extrema containing the area surplus delta S = S-(Sr+ 2 sigma r) are cut off by two straight lines to locate significant regions. The difference (S-Sr) given in standard deviation units sigma r is believed to be the amino acid substitution overall irregularity along the homologous protein sequences OI = (S-Sr)/sigma r. The significant conservative and variable regions of six homologous sequence families (phospholipase A2, cytochromes b, alpha-subunits of Na,K-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre and human rhodopsins) were identified. It was shown that for artificial homologous protein sequences derived by k-fold lengthening of natural protein sequences, the OI value rises as square root of k. To compare the degree of substitution irregularity in homologous protein sequence families of different lengths L the value of standard substitution overall irregularity for L = 250 is proposed.

Amino Acid Sequence↗
PubMed2003

The compositional adjustment of amino acid substitution matrices.

Amino acid substitution matrices are central to protein-comparison methods. In most commonly used matrices, the substitution scores take a log-odds form, involving the ratio of "target" to "background" frequencies derived from large, carefully curated sets of protein alignments. However, such matrices often are used to compare protein sequences with amino acid compositions that differ markedly from the background frequencies used for the construction of the matrices. Of course, the target frequencies should be adjusted in such cases, but the lack of an appropriate way to do this has been a long-standing problem. This article shows that if one demands consistency between target and background frequencies, then a log-odds substitution matrix implies a unique set of target and background frequencies as well as a unique scale. Standard substitution matrices therefore are truly appropriate only for the comparison of proteins with standard amino acid composition. Accordingly, we present and evaluate a rationale for transforming the target frequencies implicit in a standard matrix to frequencies appropriate for a nonstandard context. This rationale yields asymmetric matrices for the comparison of proteins with divergent compositions. Earlier approaches are unable to deal with this case in a fully consistent manner. Composition-specific substitution matrix adjustment is shown to be of utility for comparing compositionally biased proteins, including those of organisms with nucleotide-biased, and therefore codon-biased, genomes or isochores.

Amino Acid Sequence↗
PubMed1990

Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitutions.

Four amino acid substitutions (aa 176, 235, 266, and 268) have been found between the coding regions of cDNAs for histo-blood group A and B transferases (Yamamoto F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990) Nature 345, 229-233). Here we establish the basis of differential affinity of these glycosyltransferases to nucleotide-sugar (UDP-GalNAc or UDP-Gal). On the basis of gene reconstruction experiments and studies of expression in DNA transfected HeLa cells, the third as well as the fourth aa substitutions (leucine and glycine in A and methionine and alanine in B), which were calculated to modify flexibility of the protein, were found to be crucial in determining nucleotide-sugar specificity. The second substitution (glycine in A and serine in B) also affects the specificity. We have also created new enzymes which catalyze the transfer of both GalNAc and Gal, and may provide an explanation of the rare cis-AB phenotype.

ABO Blood-Group System↗
PubMed2003

[Detection of novel nucleotide substitutions resulting amino acid substitutions in TEM type beta-lactamase gene isolated from a clinically isolated extended spectrum beta-lactamases (ESBLs) productive Escherichia coli].

We screened Escherichia coli (E. coli) from specimens submitted to the Showa University hospital clinical laboratory that showed an advanced resistance to third generation cephems (Cefotaxime or Ceftazidime), as candidate extended spectrum beta-lactamases(ESBLs) producing strains. Among the candidates, two strains showed the characteristics of class A ESBLs in their susceptibility to Cefmetazole, a second generation cephem, and their resistance was inhibited by the addition of clavulanic acid. Further, we detected the TEM-type gene in one of the two E. coli strains. By determining the nucleotide sequence of the whole coding region of the TEM gene, two nucleotide substitutions with amino acid substitutions 82Val-->Ile, and 182Ala-->Val were identified.

Adult↗
PubMed1990

Deciphering the message in protein sequences: tolerance to amino acid substitutions.

An amino acid sequence encodes a message that determines the shape and function of a protein. This message is highly degenerate in that many different sequences can code for proteins with essentially the same structure and activity. Comparison of different sequences with similar messages can reveal key features of the code and improve understanding of how a protein folds and how it performs its function.

Amino Acid Sequence↗
PubMed1993

The nature and spatial distribution of amino acid substitutions conferring resistance to neutralizing monoclonal antibodies in human rhinovirus type 2.

A total of 38 neutralization escape mutant viruses have been selected from a cloned stock of human rhinovirus serotype 2 (HRV-2), using either of two monoclonal antibodies (MAbs) which recognize overlapping epitopes as judged by competition binding. The mutant viruses were analysed for their sensitivity to a panel of antiviral MAbs by antibody binding and virus neutralization assays. The position and nature of the selected mutations was determined by sequencing of the virus RNAs, and the location of the substituted amino acids on the three-dimensional structure of the virus predicted from the co-ordinates determined for the closely related HRV-1A. Escape from neutralization could be attributed to single amino acid substitutions in all but one case, which had a deletion of four amino acids. In all cases in which the same mutation was found more than once, these mutations were transitions. The ratio of transition to transversion mutations was about 5:1 overall or about 1.7:1 if only unique substitutions are considered. Each antibody selected for a discrete cluster of mutations and the area of these clusters was considerably less than that determined to be in contact with antibodies from X-ray crystallographic analyses of antibody/protein complexes. One mutation did not occur within the cluster of others selected with the same antibody. This substitution occurred at the base of a small loop and may cause conformational changes at the virus surface.

Amino Acids↗
PubMed2004

Crystal structures of the group II chaperonin from Thermococcus strain KS-1: steric hindrance by the substituted amino acid, and inter-subunit rearrangement between two crystal forms.

The crystal structures of the group II chaperonins consisting of the alpha subunit with amino acid substitutions of G65C and/or I125T from the hyperthermophilic archaeum Thermococcus strain KS-1 were determined. These mutants have been shown to be active in ATP hydrolysis but inactive in protein folding. The structures were shown to be double-ring hexadecamers in an extremely closed form, which was consistent with the crystal structure of native alpha8beta8-chaperonin from Thermoplasma acidophilum. Comparisons of the present structures with the atomic structures of the GroEL14-GroES7-(ADP)7 complex revealed that the deficiency in protein-folding activity with the G65C amino acid substitution is caused by the steric hindrance of the local conformational change in an equatorial domain. We concluded that this mutant chaperonin with G65C substitution is deprived of the smooth conformational change in the refolding-reaction cycle. We obtained a new form of crystal with a distinct space group at a lower concentration of sulfate ion in the presence of nucleotide. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion. Such subunit rotation has never been characterized in group II chaperonins. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion.

Adenosine Triphosphate↗
PubMed2006

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution.

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.

Amino Acid Substitution↗
PubMed1993

Nonpolar amino acid substitutions of potential cation binding residues glu-955 and glu-956 of the rat alpha 1 isoform of Na+, K(+)-ATPase.

Side chain oxygens are critical in the binding of cations by several macromolecules, and chemical labeling studies suggest that the carboxyl oxygens of E953 of the pig kidney Na, K-ATPase are essential for Na+ and K+ ligation. An adjacent residue, E954, is highly conserved and may also be important in cation binding. Replacement of the corresponding glutamates in the rat alpha 1 isoform (E955 and E956) with glutamines or aspartic acids has only a modest effect on enzyme activity, but both substituted amino acids contain at least one side chain oxygen, and may fulfill the role of the wild-type glutamates in binding cations. Since substitutions of amino acids lacking an oxygenated side chain have not been made at positions 955 and 956, nonpolar amino acid replacements (E955A, E956A, E955A-E956A, and E955L-E956L) were made, and the effects of the substitutions on the cation dependence properties of the mutant enzymes were examined. The substitutions were made using site-directed mutagenesis of the rat alpha 1 cDNA (which encodes a ouabain-resistant alpha subunit), followed by transfection of wild-type and mutant cDNAs into ouabain-sensitive HeLa cells, with subsequent expression of the altered proteins. Transfection with all cDNA constructs resulted in the ouabain resistance of transfected HeLa cells, demonstrating that the modified Na, K-ATPase in each case was functional. Ouabain resistance of Na, K-ATPase activity was increased 1,000-fold in microsomal membranes isolated from cells transfected with wild-type rat alpha 1 (WT) and all mutant cDNA constructs, compared to activity in membranes prepared from untransfected HeLa cells. This confirmed the expression of a ouabain-resistant enzyme in all transfected cell lines, and the enhanced ouabain resistance permitted the study of the exogenous, expressed Na, K-ATPase in the presence of 5.0 microM ouabain. Cation stimulation of exogenous Na, K-ATPase activity was not affected by the E955A substitution and only slightly by the E956A replacement (K0.5(Na+) of 3.40 +/- 0.21, 3.30 +/- 0.22, and 6.60 +/- 0.55 mM NaCl for WT, E955A and E956A, respectively; K0.5(K+) of 0.78 +/- 0.01, 0.74 +/- 0.10, and 0.56 +/- 0.11 mM KCl). Even doubly substituted enzymes had only mild alterations in cation dependence properties (K0.5(Na+) of 7.54 +/- 0.23, 7.14 +/- 0.10 mM NaCl for E955A-E956A, E955L-E956L, respectively; K0.5(K+) of 0.43 +/- 0.13, 1.83 +/- 0.13 mM KCl). The results demonstrate that there are no requirements for an oxygenated side chain at positions 955 and 956 for normal or nearly normal cation dependence.

Animals↗
PubMed1992

Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen.

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type.

Amino Acid Sequence↗
PubMed1978

Nonrandom amino acid substitution and estimation of the number of nucleotide substitutions in evolution.

A method of estimating the number of nucleotide substitutions from amino acid sequence data is developed by using Dayhoff's mutation probability matrix. This method takes into account the effect of nonrandom amino acid substitutions and gives an estimate which is similar to the value obtained by Fitch's counting method, but larger than the estimate obtained under the assumption of random substitutions (Jukes and Cantor's formula). Computer simulations based on Dayhoff's mutation probability matrix have suggested that Jukes and Holmquist's method of estimating the number of nucleotide substitutions gives an overestimate when amino acid substitution is not random and the variance of the estimate is generally very large. It is also shown that when the number of nucleotide substitutions is small, this method tends to give an overestimate even when amino acid substitution is purely at random.

Amino Acid Sequence↗
PubMed1998

Biophysical, biochemical, and physiological characterization of Chlamydomonas reinhardtii mutants with amino acid substitutions at the Ala251 residue in the D1 protein that result in varying levels of photosynthetic competence.

The QB binding site of the D1 reaction center protein, located within a stromal loop between transmembrane helices IV and V formed by residues Ile219 to Leu272, is essential for photosynthetic electron transport through photosystem II (PSII). We have examined the function of the highly conserved Ala251 D1 residue in this domain in chloroplast transformants of Chlamydomonas reinhardtii and found that Arg, Asp, Gln, Glu, and His substitutions are nonphotosynthetic, whereas Cys, Ser, Pro, Gly, Ile, Val, and Leu substitutions show various alterations in D1 turnover, photosynthesis, and photoautotrophic growth. The latter mutations reduce the rate of QA to QB electron transfer, but this is not necessarily rate-limiting for photoautotrophic growth. The Cys mutant divides and evolves O2 at wild type rates, although it has slightly higher rates of D1 synthesis and turnover and reduced electron transfer between QA and QB. O2 evolution, D1 synthesis, and accumulation in the Ser, Pro, and Gly mutants in high light is reduced, but photoautotrophic growth rate is not affected. In contrast, the Ile, Val, and Leu mutants are impaired in photoautotrophic growth and photosynthesis in both low and high light and have elevated rates of D1 synthesis and degradation, but D1 accumulation is normal. While rates of synthesis/degradation of the D1 protein are not necessarily correlated with alterations in specific parameters of PSII function in these mutants, bulkiness of the substituted amino acids is highly correlated with the dissociation constant for QB in the seven mutants examined. These observations imply that the Ala251 residue plays a key role in D1 protein.

Alanine↗