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At least 1,387 records · Page 77Linked to original sources
PubMed1996

Structure of a loop-deleted variant of 3-isopropylmalate dehydrogenase from Thermus thermophilus: an internal reprieve tolerance mechanism.

A loop-deleted mutant form of 3-isopropylmalate dehydrogenase from Thermus thermophilus was constructed to investigate the relationship between the flexibility of the structure and the thermostability of the enzyme. The structure of the mutant enzyme was determined by X-ray crystallography and was found to be almost the same as that of the native enzyme with a reduced temperature factor. Although the mutant protein had lost the flexible loop, its function and thermostability had remained unchanged. This phenomenon can be explained by an internal reprieve tolerance mechanism.

Journal Article↗
PubMed1998

Bovine hemoglobin alpha-globin chain polymorphism: primary structure determination of two new genetic variants by mass spectrometry and amino acid sequencing.

The present work describes the biochemical procedures used to identify the cause of a quantitative and qualitative hemoglobin polymorphism found in Podolian cattle. First, to analyze the different phenotypes, isoelectric focusing (IEF) of hemoglobins and RP-HPLC of globin chains was carried out; secondly, to determine accurately the globin molecular masses, electrospray mass spectrometry was performed and finally to check the entire amino acid sequences of the proteins, several enzymatic digests were analyzed by fast atom bombardment mass spectrometry (FAB-MS) and Edman degradation procedure. As to the qualitative polymorphism, the results of RP-HPLC show the presence of two alpha-globin variants to which the extensive mass spectrometric analysis attributed a molecular mass of 15,026.47 +/- 0.44 Da and 15,079.86 +/- 0.66 Da and whose respective primary structure differed from that of the common alpha-globin chain in the amino acid substitution Asn-->Ser at position 131 and the other in the replacement of the histidine residue at position 89 with tyrosine. As to the quantitative polymorphism, on the basis of the expression gradient found out in the duplicated alpha genes of several mammals, we conceive that the alpha 89 His-->Tyr is an allelic form of the I alpha gene while the alpha 131Asn-->Ser is an allelic form of the II alpha gene.

Amino Acid Sequence↗
PubMed2003

Structural analysis, fatty acid and thyroxine binding properties of Vancouver and Naskapi variants of human serum albumin.

OBJECTIVES: To purify and structurally identify two albumin variants found in the Canadian population of native Amerindian origin. To assess the ability of variant albumins to bind lauric acid and L-thyroxine. METHODS: The structural characterization of the alloalbumins was performed by conventional protein chemistry methods and by mass spectrometric analysis. Lauric acid and L-thyroxine affinities to variant albumins were assessed by kinetic dialysis and equilibrium dialysis techniques, respectively. RESULTS: The sequence investigations proved the two variants to be albumin Naskapi [372Lys --> Glu] and albumin Vancouver [501Glu --> Lys], respectively. Among the carriers of albumin Naskapi, we found a rare case of homozygosity. Furthermore, this is the first reported case of the 501Glu-->Lys mutation in the native North American population. Scatchard plot analysis revealed that the association constants for lauric acid and L-thyroxine to the two variants were indistinguishable from the endogenous form of albumin. CONCLUSION: We show that albumin variants Vancouver and Naskapi have normal fatty acid and L-thyroxine binding capabilities. These findings support the assumption that bisalbuminemias associated with these albumin variants are benign conditions.

British Columbia↗
PubMed2005

Ca2+ signalling, voltage-gated Ca2+ channels and praziquantel in flatworm neuromusculature.

Transient changes in calcium (Ca2+) levels regulate a wide variety of cellular processes, and cells employ both intracellular and extracellular sources of Ca2+ for signalling. Praziquantel, the drug of choice against schistosomiasis, disrupts Ca2+ homeostasis in adult worms. This review will focus on voltage-gated Ca2+ channels, which regulate levels of intracellular Ca2+ by coupling membrane depolarization to entry of extracellular Ca2+. Ca2+ channels are members of the ion channel superfamily and represent essential components of neurons, muscles and other excitable cells. Ca2+ channels are membrane protein complexes in which the pore-forming alpha1 subunit is modulated by auxiliary subunits such as beta and alpha2delta. Schistosomes express two Ca2+ channel beta subunit subtypes: a conventional subtype similar to beta subunits found in other vertebrates and invertebrates and a novel variant subtype with unusual structural and functional properties. The variant schistosome beta subunit confers praziquantel sensitivity to an otherwise praziquantel-insensitive mammalian Ca2+ channel, implicating it as a mediator of praziquantel action.

Animals↗
PubMed2004

B beta Glu397 and B beta Asp398 but not B beta Asp432 are required for "B:b" interactions.

We synthesized three fibrinogen variants, BbetaE397A, BbetaD398A, and BbetaD432A, with substitutions at positions identified in crystallographic studies as critical for binding the "B" peptide, Gly-His-Arg-Pro-amide (GHRPam), to the "b" polymerization site. We examined thrombin- and batroxobin-catalyzed polymerization by turbidity measurements and found that BbetaE397A and BbetaD398A were impaired while BbetaD432A was normal. Changes in polymerization as a function of calcium were similar for variant and normal fibrinogens. We determined crystal structures of fragment D from the variant BbetaD398A in the absence and presence of GHRPam. In the absence of peptide, the structure showed that the alanine substitution altered only specific local interactions, as alignment of the variant structure with the analogous normal structure resulted in an RMSD of 0.53 A over all atoms. The structure also showed reduced occupancy of the beta2 calcium-binding site that includes the side chain carbonyl of BbetaD398, suggesting that calcium was not bound at this site in our polymerization studies. In the presence of peptide, the structure showed that GHRPam was not bound in the "b" site and the conformational changes associated with peptide binding to normal fragment D did not occur. This structure also showed GHRPam bound in the "a" polymerization site, although in two different conformations. Calcium binding was associated with only one of these conformations, suggesting that calcium binding to the gamma2-site and an alternative peptide conformation were induced by crystal packing. We conclude that BbetaE397 and BbetaD398 are essential for the "B:b" interaction, while BbetaD432 is not.

Alanine↗
PubMed1996

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

2,2'-Dipyridyl↗
PubMed1979

Structural difference between the normal PiM1 and the common PiM2 variant of human alpha 1-antitrypsin.

The common PiM2 variant of human alpha 1-antitrypsin (alpha 1-AT) which can be distinguished from the wild type PiM1 by isoelectric focusing (IEF) in a narrow pH gradient, was purified to homogeneity from plasma of a homozygous PiM2/PiM2 subject. The specific trypsin inhibitory activity and the amino acid and carbohydrate composition of the normal PiM1 and the variant PiM2 are very similar. The structural difference between the normal and the variant inhibitors was elucidated by peptide mapping of their tryptic digests. An amino acid substitution of glutamic acid in the normal inhibitor by aspartic acid in the variant inhibitor was found. The same amino acid substitution was found in PiMN, which was presumed to be identical to PiM2 based on their IEF patterns.

Amino Acid Sequence↗
PubMed2004

Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains.

The pleckstrin homology (PH) domains of the homologous proteins Grp1 (general receptor for phosphoinositides), ARNO (Arf nucleotide binding site opener), and Cytohesin-1 bind phosphatidylinositol (PtdIns) 3,4,5-trisphosphate with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the beta1/beta2 loop exhibit dual specificity for PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2). The structural basis for this dramatic specificity switch is not apparent from the known modes of phosphoinositide recognition. Here, we report crystal structures for dual specificity variants of the Grp1 and ARNO PH domains in either the unliganded form or in complex with the head groups of PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). Loss of contacts with the beta1/beta2 loop with no significant change in head group orientation accounts for the significant decrease in PtdIns(3,4,5)P(3) affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PtdIns(4,5)P(2) is explained by a novel binding mode, in which the glycine insertion alleviates unfavorable interactions with the beta1/beta2 loop. These observations are supported by a systematic mutational analysis of the determinants of phosphoinositide recognition.

Alternative Splicing↗
PubMed1988

[Anatomo-anthropological typology of the jaws in man].

Basing on the morphological signs selected in the morphologically contrast samples of the facial skeleton, 27 variants of the upper and lower jaws have been obtained for each, characterizing interconnection and interdependence of several morphological signs. Into each of these variants people of various races and sex, with massive or gracile masticatory apparatus can get. However, the reduction degree, directness, temps and successiveness of reorganizations together with exogenic influence within the limits of the normal reaction in various parts of the masticatory apparatus of the individuals, that belong to the same variant, is identical. Thus, an anatomo-anthropological typology of the dento-maxillary apparatus is obtained, owing to which it is possible to study and compare: 1--individual morphological peculiarities, 2--regional and ethnic norms in morphology of the jaws, 3--within the 27 variants of the structure selected it is possible to study functional, biochemical and other factors. The results demonstrate, that 27 morphological variants of the jaw structure can serve as anatomical marker for distinguishing 27 risk groups for caries.

Adolescent↗
PubMed2001

Phosphorylation of histone variant regions in chromatin: unlocking the linker?

Histone variants illuminate the behavior of chromatin through their unique structures and patterns of postsynthetic modification. This review examines the literature on heteromorphous histone structures in chromatin, structures that are primary targets for histone kinases and phosphatases in vivo. Special attention is paid to certain well-studied experimental systems: mammalian culture cells, chicken erythrocytes, sea urchin sperm, wheat sprouts, Tetrahymena, and budding yeast. A common theme emerges from these studies. Specialized, highly basic structures in histone variants promote chromatin condensation in a variety of developmental situations. Before, and sometimes after condensed chromatin is formed, the chromatin is rendered soluble by phosphorylation of the heteromorphous regions, preventing their interaction with linker DNA. A simple structural model accounting for histone variation and phosphorylation is presented.

Amino Acid Sequence↗
PubMed2005

Variations in Helicobacter pylori lipopolysaccharide to evade the innate immune component surfactant protein D.

Helicobacter pylori is a common and persistent human pathogen of the gastric mucosa. Surfactant protein D (SP-D), a component of innate immunity, is expressed in the human gastric mucosa and is capable of aggregating H. pylori. Wide variation in the SP-D binding affinity to H. pylori has been observed in clinical isolates and laboratory-adapted strains. The aim of this study was to reveal potential mechanisms responsible for evading SP-D binding and establishing persistent infection. An escape variant, J178V, was generated in vitro, and the lipopolysaccharide (LPS) structure of the variant was compared to that of the parental strain, J178. The genetic basis for structural variation was explored by sequencing LPS biosynthesis genes. SP-D binding to clinical isolates was demonstrated by fluorescence-activated cell sorter analyses. Here, we show that H. pylori evades SP-D binding through phase variation in lipopolysaccharide. This phenomenon is linked to changes in the fucosylation of the O chain, which was concomitant with slipped-strand mispairing in a poly(C) tract of the fucosyltransferase A (fucT1) gene. SP-D binding organisms are predominant in mucus in vivo (P = 0.02), suggesting that SP-D facilitates physical elimination. Phase variation to evade SP-D contributes to the persistence of this common gastric pathogen.

Carbohydrate Sequence↗
PubMed2005

Molecular dynamics study of water penetration in staphylococcal nuclease.

The ionization properties of Lys and Glu residues buried in the hydrophobic core of staphylococcal nuclease (SN) suggest that the interior of this protein behaves as a highly polarizable medium with an apparent dielectric constant near 10. This has been rationalized previously in terms of localized conformational relaxation concomitant with the ionization of the internal residue, and with contributions by internal water molecules. Paradoxically, the crystal structure of the SN V66E variant shows internal water molecules and the structure of the V66K variant does not. To assess the structural and dynamical character of interior water molecules in SN, a series of 10-ns-long molecular dynamics (MD) simulations was performed with wild-type SN, and with the V66E and V66K variants with Glu66 and Lys66 in the neutral form. Internal water molecules were identified based on their coordination state and characterized in terms of their residence times, average location, dipole moment fluctuations, hydrogen bonding interactions, and interaction energies. The locations of the water molecules that have residence times of several nanoseconds and display small mean-square displacements agree well with the locations of crystallographically observed water molecules. Additional, relatively disordered water molecules that are not observed crystallographically were found in internal hydrophobic locations. All of the interior water molecules that were analyzed in detail displayed a distribution of interaction energies with higher mean value and narrower width than a bulk water molecule. This underscores the importance of protein dynamics for hydration of the protein interior. Further analysis of the MD trajectories revealed that the fluctuations in the protein structure (especially the loop elements) can strongly influence protein hydration by changing the patterns or strengths of hydrogen bonding interactions between water molecules and the protein. To investigate the dynamical response of the protein to burial of charged groups in the protein interior, MD simulations were performed with Glu66 and Lys66 in the charged state. Overall, the MD simulations suggest that a conformational change rather than internal water molecules is the dominant determinant of the high apparent polarizability of the protein interior.

Algorithms↗
PubMed1984

Human hypoxanthine-guanine phosphoribosyltransferase. Structural alteration in a dysfunctional enzyme variant (HPRTMunich) isolated from a patient with gout.

HPRTMunich is a mutant form of human hypoxanthine-guanine phosphoribosyltransferase that was isolated from a patient who presented with gout and a partial deficiency of enzyme activity. Profound abnormalities in the catalytic function of HPRTMunich are responsible for the deficiency of enzyme activity in vivo. Tryptic peptides of HPRTMunich were mapped by reverse phase high pressure liquid chromatography in an attempt to define the precise abnormality in its primary structure. Sequence analysis of aberrant peptides localized the structural alteration in HPRTMunich to residue 103. Several additional findings suggest that the mutation in this variant is most likely a serine to arginine substitution at residue 103. This amino acid substitution lies within the putative hypoxanthine-binding site of human hypoxanthine-guanine phosphoribosyltransferase possibly explaining its selective effect on intrinsic enzyme activity and binding of hypoxanthine.

Amino Acid Sequence↗
PubMed1976

Type 1 and 2 synaptic junctions: differences in distribution of concanavalin A binding sites and stability of the junctional adhesion.

Two classes of synaptosomes have been distinguished in subcellular fractions from rat forebrain. They differ in morphology and distribution of concanavalin A binding sites. Morphological examination suggests that the two classes correspond to the type 1 (excitatory) and type 2 (inhibitory) synapses of intact brain tissue. Under the conditions of incubation employed in this study, type 1 synaptosomes show splitting of the synaptic junctions, but the type 2 junctions are apparently stable. The chemical differences illustrated by the differences in concanavalin A binding sites suggest that type 1 and type 2 synaptic junctions differ fundamentally in supramolecular structure rather than being superficial variants of basically similar structures.

Animals↗
PubMed2006

Ce53Fe12S90X3 (X = Cl, Br, I): the first rare-earth transition-metal sulfide halides.

The compounds Ce53Fe12S90X3 (X = Cl, Br, I), which represent the first examples of rare-earth transition-metal sulfide halides, were prepared using the reactive-flux method, through reaction of Ce2S3, FeS, or Fe and S in a CeX3 flux at 1320 K. Their structures were determined by single-crystal X-ray diffraction. The compounds are isostructural, crystallizing in the trigonal space group Rm with Z = 1 [Ce53Fe12S90Cl3, a = 13.9094(9) A, c = 21.604(2) A, V = 3619.7(4) A3; Ce(53)Fe(12)S(90)Br(3), a = 13.916(1) A, c = 21.824(2) A, V = 3660.0(5) A3; Ce53Fe12S90I3, a = 13.863(3) A, c = 21.944(6) A, V = 3652(2) A3]. The structure adopted is a stuffed variant of the La52Fe12S90 structure type. Fe2S9 dimers of face-sharing octahedra are linked by face- and vertex-sharing capped CeS6 trigonal prisms, forming a three-dimensional framework containing cuboctahedral cavities of two sizes. The smaller cavities accommodate alternative sites for disordered cerium atoms. The larger cavities, which remain empty in the parent structure, are filled by halogen atoms in Ce53Fe12S90X3. Alternatively, the structure can be described as a 9-fold superstructure of the Mn5Si3 structure type (P6(3)/mcm), with a = a' and c = 3c'. Temperature-dependent magnetic susceptibility measurements suggest that Ce53Fe12S90I3 may order antiferromagnetically at low temperatures.

Journal Article↗
PubMed2001

Structure, properties, and bonding of ZrTe (MnP type), a low-symmetry, high-temperature modification of ZrTe (WC type).

ZrTe (MnP) was synthesized by high-temperature methods at 1570 K in Ta ampules. The structure of the telluride was determined by means of single-crystal X-ray diffraction to be orthorhombic, Pnma (No. 62), Z = 4, Pearson Symbol oP8, a = 739.15(15) pm, b = 377.23(8) pm, c = 694.34(14) pm. The orthorhombic MnP type structure is a distorted variant of the NiAs type structure with pronounced metal-metal zigzag chains. Zigzag chains are typical for phases with a d(2) metal atom electron configuration. According to extended Hückel calculations, the homonuclear interactions in the zigzag chains make up for 2/3 of the Zr-Zr interactions in ZrTe (MnP) and contribute decisively to the stability of the structure. The emergence of the distorted MnP type structure instead of the high-symmetry NiAs type ZrTe at high temperatures can be understood as the result of an optimization of homonuclear Zr-Zr interactions arising from states close to the Fermi level. The hexagonal WC type ZrTe transforms above 1438 +/- 5 K into ZrTe (MnP) (DeltaH = 8.3 +/- 1.0 kJ mol(-1)). The phase transition is reversible, although at room-temperature the reverse reaction is kinetically inhibited. Zr5Te4 and Zr5Te6 are the phases next to ZrTe. ZrTe (MnP) exhibits temperature-independent paramagnetic properties (chi(mol) = 0.14 x 10(-3) cm(3) mol(-1)), as typical for a metallic conductor. Resistivity measurements on ZrTe (MnP) imply metallic behavior.

Journal Article↗
PubMed2003

Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli.

The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.

Amino Acid Substitution↗
PubMed2025

Engineered histones reshape chromatin in human cells.

Histone proteins and their variants have been found to play crucial and specialized roles in chromatin organization and the regulation of downstream gene expression; however, the relationship between histone sequence and its effect on chromatin organization remains poorly understood, limiting our functional understanding of sequence variation between distinct subtypes and across evolution and frustrating efforts to rationally design synthetic histones that can be used to engineer specified cell states. Here, we make the first advance towards engineered histone-driven chromatin organization. By expressing libraries of sequence variants of core histones in human cells, we identify variants that dominantly modulate chromatin structure. We further interrogate variants using a combination of imaging, proteomics, and genomics to reveal both cis and trans-acting mechanisms of effect. Functional screening with transcription factor libraries identifies transcriptional programs that are facilitated by engineered histone expression. Double mutation screens combined with protein language models allow us to learn sequence-to-function patterns and design synthetic histone proteins optimized to drive specific chromatin states. This work establishes a foundation for the high-throughput evaluation and engineering of chromatin-associated proteins and positions histones as tunable nodes for understanding and modulating mesoscale chromatin organization.

Journal Article↗