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At least 865 records · Page 48Linked to original sources
PubMed1997

Structures of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease.

Cys319 is located on a mobile flap covering the active site of Klebsiella aerogenes urease but does not play an essential role in catalysis. Four urease variants altered at position C319 range from having high activity (C319A) to no measurable activity (C319Y), indicating Cys is not required at this position, but its presence is highly influential [Martin, P. R., & Hausinger, R. P. (1992) J. Biol. Chem. 267, 20024-20027]. Here, we present 2.0 A resolution crystal structures of C319A, C319S, C319D, and C319Y proteins and the C319A variant inhibited by acetohydroxamic acid. These structures show changes in the hydration of the active site nickel ions and in the position and flexibility of the active site flap. The C319Y protein exhibits an alternate conformation of the flap, explaining its lack of activity. The changes in hydration and conformation suggest that there are suboptimal protein-solvent and protein-protein interactions in the empty urease active site which contribute to urease catalysis. Specifically, we hypothesize that the suboptimal interactions may provide a significant source of substrate binding energy, and such hidden energy may be a common phenomenon for enzymes that contain mobile active site loops and undergo an induced fit. The acetohydroxamic acid-bound structure reveals a chelate interaction similar to those seen in other metalloenzymes and in a small molecule nickel complex. The inhibitor binding mode supports the proposed mode of urea binding. We complement these structural studies with extended functional studies of C319A urease to show that it has enhanced stability and resistance to inhibition by buffers containing nickel ions. The near wild-type activity and enhanced stability of the C319A variant make it useful for further studies of urease structure-function relationships.

Amino Acid Sequence↗
PubMed1980

Structural analysis of variant and invariant genes in trypanosomes.

Variant surface antigens have been purified from variants of a newly developed serodeme of Trypanosoma brucei 164, and found to be distinct by immunological and molecular criteria. cDNA clones containing variant and invariant gene sequences were isolated by using RNA from different variants and cloned DNAs from other systems as probes. These trypanosome and heterologous cloned DNAs were used as probes of trypanosome gene structure and activity. Regulation of variant antigen gene expression is at the level of transcription, and the variant antigen genomic sequences differ in their arrangement between the variants. Chick alpha- and beta-tubulin sequences hybridize to trypanosome-tubulin gene sequences, while chick beta-actin sequences do not. A 1650 bp alpha-tubulin trypanosome cDNA has been isolated. The trypanosome alpha- and beta-tubulin sequences appear to be clustered in the genome. Sequence rearrangement of tubulin genes does not occur during antigenic variation.

Animals↗
PubMed2002

Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential.

Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.

Azotobacter vinelandii↗
PubMed1998

The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).

Alanine↗
PubMed1998

Preparation and kinetic characterization of a series of betaW37 variants of human hemoglobin A: evidence for high-affinity T quaternary structures.

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.

Amino Acid Substitution↗
PubMed2004

Novel inhibitors of bacterial protein synthesis: structure-activity relationships for 1,8-naphthyridine derivatives incorporating position 3 and 4 variants.

Structure-activity relationships for a recently discovered novel ribosome inhibitor (NRI) class of antibacterials were investigated. Preliminary efforts to optimize protein synthesis inhibitory activity of the series through modification of positions 3 and 4 of the naphthyridone lead template resulted in the identification of several biochemically potent analogues. A lack of corresponding whole cell antibacterial activity is thought to be a consequence of poor cellular penetration as evidenced by the enhancement of activity observed for a lead analogue tested in the presence of a cell permeabilizing agent.

Anti-Bacterial Agents↗
PubMed2005

From DNA to fitness differences: sequences and structures of adaptive variants of Colias phosphoglucose isomerase (PGI).

Colias eurytheme butterflies display extensive allozyme polymorphism in the enzyme phosphoglucose isomerase (PGI). Earlier studies on biochemical and fitness effects of these genotypes found evidence of strong natural selection maintaining this polymorphism in the wild. Here we analyze the molecular features of this polymorphism by sequencing multiple alleles and modeling their structures. PGI is a dimer with rotational symmetry. Each monomer provides a critical residue to the other monomer's catalytic center. Sequenced alleles differ at multiple amino acid positions, including cryptic charge-neutral variation, but most consistent differences among the electromorph alleles are at the charge-changing amino acid sites. Principal candidate sites of selection, identified by structural and functional analyses and by their variants' population frequencies, occur in interpenetrating loops across the interface between monomers, where they may alter subunit interactions and catalytic center geometry. Comparison to a second (and basal) species, Colias meadii, also polymorphic for PGI under natural selection, reveals one fixed amino acid difference between their PGIs, which is located in the interpenetrating loop and accompanies functional differences among their variants. We also study nucleotide variability among the PGI alleles, comparing these data to similar data from another glycolytic enzyme gene, glyceraldehyde-3-phosphate dehydrogenase. Despite extensive nonsynonymous and synonymous polymorphism at PGI in each species, the only base changes fixed between species are the two causing the amino acid replacement; this absence of synonymous fixation yields a significant McDonald-Kreitman test. Analyses of these data suggest historical population expansion. Positive peaks of Tajima's D statistic, representing regions of neutral "hitchhiking," are found around the principal candidate sites of selection. This study provides novel views of molecular-structural mechanisms, and beginnings of historical evidence, for a long-persistent balanced enzyme polymorphism at PGI in these and perhaps other species.

Alleles↗
PubMed1992

Structure of scorpion toxin variant-3 at 1.2 A resolution.

The crystal structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been refined at 1.2 A resolution using restrained least-squares. The final model includes 492 non-hydrogen protein atoms, 453 protein hydrogen atoms, eight 2-methyl-2,4-pentanediol (MPD) solvent atoms, and 125 water oxygen atoms. The variant-3 protein model geometry deviates from ideal bond lengths by 0.024 A and from ideal angles by 3.6 degrees. The crystallographic R-factor for structure factors calculated from the final model is 0.192 for 17,706 unique reflections between 10.0 to 1.2 A. A comparison between the models of the initial 1.8 A and the 1.2 A refinement shows a new arrangement of the previously poorly defined residues 31 to 34. Multiple conformations are observed for four cysteine residues and an MPD oxygen atom. The electron density indicates that disulfide bonds between Cys12 and Cys65 and between Cys29 and Cys48 have two distinct side-chain conformations. A molecule of MPD bridges neighboring protein molecules in the crystal lattice, and both MPD enantiomers are present in the crystal. A total of 125 water molecules per molecule of protein are included in the final model with B-values ranging from 11 to 52 A2 and occupancies from unity down to 0.4. Comparisons between the 1.2 A and 1.8 A models, including the bound water structure and crystal packing contacts, are emphasized.

Amino Acid Sequence↗
PubMed1998

The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications.

We have solved the structure of two active-site variants of soluble inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85 and 2.15 A and R-factors of 19.5% and 18.3%, respectively. In the R78K variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction between P1 and Arg78, and solution data showing a decrease in P1 affinity in the variant. The structure explains why the mutation affects P1 and pyrophosphate binding much more than would be expected by the loss of one hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an interaction with P1. The R78K variant also provides the first direct evidence that the low-affinity phosphate group (P2) can adopt the structure that we believe is the immediate product of hydrolysis, with one of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase is, indeed, the attacking nucleophile. The D117E variant structure likewise supports our model of catalysis, as the Glu117 variant carboxylate group is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile is replaced by a carboxylate co-ordinated to two metal ions. Alternative confirmations of Glu117 may allow Wat1 to be present but at much reduced occupancy, explaining why the pKa of the nucleophile increases by three pH units, even though there is relatively little distortion of the active site. These new structures, together with parallel functional studies measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is considered unimportant for hydrolysis. They thus support the notion that PPase shares mechanistic similarity with the "two-metal ion" mechanism of polymerases.

Amino Acid Sequence↗
PubMed1999

Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, growth with a non-fermentable carbon source requires co-ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source-responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO2TAD) and/or a carbon source-independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE-dependent synthetic minimal promoter showed a 40-fold derepression with wild-type CAT8, an almost constitutive expression was found with a MET25-CAT8-INO2TAD fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source-dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.

Carbon↗
PubMed1988

Gamma gene promoter and enhancer structure in Seattle variant of hereditary persistence of fetal hemoglobin.

A variant of hereditary persistence of fetal hemoglobin (HPFH), first described in a patient from Seattle, was studied by structural analysis of the gamma-globin genes. A family study suggested that the determinant for this form of HPFH, in which the HbF contains both G gamma- and A gamma-globin chains, segregated with the beta S gene. No deletions or other abnormalities were detected in the fetal to adult globin gene region by genomic mapping studies. All four gamma-globin genes were isolated from a cosmid library, and allelic pairs of gamma-globin genes were distinguished by linkage to either the beta S- or beta A-globin gene. Nucleotide sequence analysis of the four gamma-globin gene promoters revealed a total of three discrepancies compared with a reference sequence, but these were judged unlikely to be the underlying determinants. Sequence analysis of the enhancer region located 3' to the A gamma-globin gene from the putative HPFH chromosome revealed three base substitutions, whereas this region was normal in the A gamma-globin gene linked to the beta A gene. These data raise the possibility that an alteration of enhancer function rather than promoter function could be the basis for this condition.

Base Sequence↗
PubMed2000

Gene encoding the catalytic subunit p110beta of human phosphatidylinositol 3-kinase: cloning, genomic structure, and screening for variants in patients with type 2 diabetes.

Phosphatidylinositol (PI) 3-kinase is a key signaling molecule in insulin-stimulated glucose transport. Therefore, we investigated the catalytic subunit p110beta, of human PI 3-kinase as a candidate gene for type 2 diabetes. Human p110beta gene was cloned from the placental genomic library. All 22 exons, intronic regions flanking the exons and 1.5 kb of the proximal/5' region of the p110beta gene, were screened for variants by single-strand conformation polymorphism analysis in 79 Finnish patients with type 2 diabetes . Allele frequencies of the variants were also determined in 77 nondiabetic control subjects. No variants were found in exons in diabetic patients. However, we identified two nucleotide polymorphisms in the proximal/5' region of the p110beta gene and a variation in the number of 2-bp repeat sequence (TA)n in intron 4. The allele frequencies did not differ between diabetic and control subjects. Our results may indicate that the catalytic subunit p110beta of PI 3-kinase plays such a fundamental role in the insulin-signaling pathway that structural variants are not likely to exist in that gene. The importance of the polymorphisms in the proximal/5' region of the p110beta gene for insulin signaling remains to be determined.

Alleles↗
PubMed1996

Structural characterization of variant forms of arylsulfatase A that associate with alcoholism.

Several electrophoretic forms of human platelet arylsulfatase A (ASA), including variant type IIIa and normal type IV(a), have been identified by nondenaturing polyacrylamide gel electrophoresis. An alcoholic population that we have analyzed is enriched in variant type IIIa compared with nonalcoholic psychiatric and normal controls. Individuals with the IIIa enzyme possess greatly reduced levels of ASA activity. To understand further the structural basis for the differences and their potential biological consequences, the nature of the ASA variant expressed by fibroblasts from different individuals was explored. The electrophoretic patterns of fibroblast ASA from the IIIa and IV(a) individuals differ in degree of phosphorylation. Furthermore, fibroblast ASA from IIIa individuals lacks an N-linked glycan found in ASA from IV(a) individuals. In addition, differences in peptide and/or posttranslational modification unrelated to the N-linked carbohydrate or phosphorylation exist between the fibroblast ASA from IIIa and IV(a) individuals. The finding that both fibroblasts and platelets exhibit related electrophoretic isoform patterns characteristic of the donor's ASA type allows for the use of fibroblasts to study the impact of ethanol on the metabolism of cells possessing different ASA types.

Alcoholism↗
PubMed1991

Purification and structural study of two albumin variants in an Irish population.

Two types of variant albumins were detected during routine electrophoresis on cellulose acetate on 34,000 sera from patients in a relatively stable Irish population. The fast type (IRE1) (relative mobility 1.05) had a heterozygote frequency of 1/3,780, and the slow type (IRE2) (relative mobility of 0.94) had a heterozygote frequency of 1/8,500. A method for purification of the two types of variants is described. Structural study of the fast variant established a single amino acid substitution 313 lysine----asparagine (313 Lys----Asn); this variant has been reported in several European populations and also in New Guinea indigenes. However, the slow variant has a new substitution, 479 glutamic acid----lysine (479 Glu----Lys). Because it appears to be uniquely Irish, the slow variant (formerly called IRE2) has been renamed albumin Dublin. Three other albumin variants most often reported in European populations (cumulative frequency only about 1/3,500) were not detected in this study. Because of the significance of albumin genetic variants for the study of protein evolution and as an aid in identification of drug-binding sites, clinical chemists are asked to be on the alert for cases of bisalbuminemia.

Adult↗
PubMed2005

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway.

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Bacterial Proteins↗
PubMed1993

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins.

The DNA sequence of bovine microsomal cytochrome b5 has been amplified from a liver cDNA library using a polymerase chain reaction. The amplified cDNA when cloned into plasmids that support the high-level production of cytochrome b5 in E.coli leads to protein overexpression and results in cell colonies bearing a strong red colouration. Using cassette mutagenesis, truncated versions of the cytochrome b5 cDNA have been made that encode the first 90 amino acid residues (Ala1-Lys90), the first 104 amino acids (Ala1-Ser104) and the complete protein (Ala1-Asn133). The location of the overexpressed cytochrome b5 within prokaryotic cells is dependent on the overall length of the protein. Expression of the Ala-Lys90 and Ala1-Ser104 variants leads to a location in the cytoplasmic phase of the bacteria whereas the whole protein, Ala1-Asn133, is found within the bacterial membrane fraction. The last 30 residues of cytochrome b5 therefore contain all of the necessary information to insert the protein into E.coli membranes. The solubility of the Ala1-Ser104 variant permits the solution structure and stability of this protein to be measured using 1- and 2-D 1H-NMR methods and electronic spectroscopy. 1-D NMR studies show that the chemical shifts of the haem and haem ligand resonances of the Ala1-Ser104 variant exhibit only very slight perturbations to their magnetic microenvironments when compared with the tryptic fragment of ferricytochrome b5. These results indicate an arrangement of residues in the haem pocket that is very similar in both the Ala1-Ser104 variant and the tryptic fragment and by 2-D NMR it is shown that this similarity extends to the conformations of the polypeptide backbone and side chains. Electronic spectroscopy of this variant shows absorbance maxima for the Soret peaks at 423 nm (reduced) and 413 nm (oxidized). From absorbance spectra the relative thermal stabilities of the Ala1-Ser104 variant and the tryptic fragment were measured. In the oxidized state the Ala1-Ser104 variant denatures in a single cooperative transition with a midpoint temperature (Tm) of 73 degrees C that is significantly higher than that of 'tryptic' ferricytochrome b5. The reduced form of the protein shows increased transition temperatures (Tm approximately 78 degrees C) reflected in the values of delta Hm, delta Sm and delta(delta G) of 420 kJ/mol, 1096 J/mol/K and 12.38 kJ/mol respectively, estimated for this variant. The increased stability of the Ala1-Ser104 variant and other recombinant forms of cytochrome b5 is correlated with the presence of additional residues at the N- and C-termini.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗