bluuebunny/crossref_metadata_2025_split huggingface dataset:
Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase
Authors: Adams Joseph A. , Taylor Susan S. 
Published: February 2009

AbstractPhosphorylation of the peptide LRRASLG by the catalytic subunit of cAMP‐dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second‐order rate constant, kcat/Kpeptide, and the maximal rate constant, kcat, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E‐ATP‐S. The effects of solvent viscosity on the steady‐state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kcat and kcat/Kpeptide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, Ea‐ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that cAMP‐dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.
Probing weakly polar interactions in cytochrome c
Authors: Auld Douglas S. , Young Gregory B. , Saunders Aleister J. , Doyle Donald F. , Betz Stephen F. , Pielak Gary J. 
Published: February 2009

AbstractTheoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur‐containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso‐1‐cytochrome c genes at positions that form a weakly polar aromatic‐aromatic interaction in the wild‐type protein. To determine if a weakly polar sulfur‐aromatic interaction replaced the aromatic‐aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cystine analog, S‐methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur‐containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur‐containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2–3 kcal mol−1 less stable than iso‐1‐cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 + 0.3 kcal mol−1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur‐containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.
Conservation of solvent‐binding sites in 10 crystal forms of T4 lysozyme
Authors: Zhang X.‐J. , Matthews B.W. 
Published: February 2009

AbstractSolvent‐binding sites were compared in 10 different crystal forms of phage T4 lysozyme that were refined using data from 2.6 Å to 1.7 Å resolution. The sample included 18 crystallographically independent lysozyme molecules.Despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20 most frequently occupied sites were conserved. Allowing for potential steric interference from neighboring molecules in the crystal lattice, this fraction increased to 79% of the sites. There was, however, no solvent‐binding site that was occupied in all 18 lysozyme molecules. A buried double site was occupied in 17 instances and 2 other internal sites were occupied 15 times. Apart from these buried sites, the most frequently occupied sites were often at the amino‐termini of α‐helices. Solvent molecules at the most conserved sites tended to have crystallographic thermal factors lower than average, but atoms with low B‐factors were not restricted to these sites.Although superficial inspection may suggest that only 50–60% (or less) of solvent‐binding sites are conserved in different crystal forms of a protein, it appears that many sites appear to be empty either because of steric interference or because the apparent occupancy of a given site can vary from crystal to crystal. The X‐ray method of identifying sites is somewhat subjective and tends to result in specification only of those solvent molecules that are well ordered and bound with high occupancy, even though there is clear evidence for solvent bound at many additional sites. Because similar sites are occupied under a variety of crystallization conditions, it seems likely that binding of solvent to such sites will be maintained in solution under physiological conditions.
A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential
Authors: Jung Heinrich , Jung Kirsten , Kaback H. Ronald 
Published: February 2009

AbstractLactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N‐ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (δμ, Sahin‐Tóth M, Kaback HR, 1993, Protein Sci 2:1024–1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315Ψ Cys permease reacts with N‐ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and β‐galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315 Ψ Cys permease is enhanced by imposition of a membrane potential (δΨ, interior negative). The results indicate that either ligand binding or ΔΨ induces a conformational change in the permease that brings the N‐terminus of helix X into an environment that is more accessible from the lipid phase.
Engineering of betabellin 14D: Disulfide‐induced folding of a β‐sheet protein
Authors: Yan Yibing , Erickson Bruce W. 
Published: February 2009

AbstractThe betabellin target structure consists of 2 32‐residue β sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14D, the disulfide‐bridged double chain. The 32‐residue nongenetic betabellin‐14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = D‐Ala, k = D‐Lys) has a palindromic pattern of polar (p), nonpolar (n), end (e), and β‐turn (t, r) residues (epnpnpnttnpnpnprrpnpnpnttnpnpnpe). Each half contains the same 14‐residue palindromic pattern (underlined). Pairs of D‐amino acid residues are used to favor formation of inverse‐common (type‐I') β turns. In water at pH 6.5, the single chain of betabellin 14S is not folded, but the disulfide‐linked betabellin 14D is folded into a stable β‐sheet structure. Thus, folding of the covalent dimer betabellin 14D is induced by formation of the single interchain disulfide bond. The binary pattern of alternating polar and nonpolar residues of its β sheets is not sufficient to induce folding. Betabellin 14D is a very water‐soluble (10 mg/mL), small (64 residues), nongenetic (12 D residues) β‐sheet protein with properties (well‐dispersed proton NMR resonances; Tm = 58 °C and δHm = 106 kcal/mol at pH 5.5) like those of a native protein structure.
Unexpected sequence similarity between nucleosidases and phosphoribosyltransferases of different specificity
Authors: Mushegian Arcady R. , Koonin Eugene V. 
Published: February 2009

AbstractAmino acid sequences of enzymes that catalyze hydrolysis or phosphorolysis of the N‐glycosidic bond in nucleosides and nucleotides (nucleosidases and phosphoribosyltransferases) were explored using computer methods for database similarity search and multiple alignment. Two new families, each including bacterial and eukaryotic enzymes, were identified. Family I consists of Escherichia coli AMP hydrolase (Amn), uridine phosphorylase (Udp), purine phosphorylase (DeoD), uncharacterized proteins from E. coli and Bacteroides uniformis, and, unexpectedly, a group of plant stress‐inducible proteins. It is hypothesized that these plant proteins have evolved from nucleosidases and may possess nucleosidase activity. The proteins in this new family contain 3 conserved motifs, one of which was found also in eukaryotic purine nucleosidases, where it corresponds to the nucleoside‐binding site. Family II is comprised of bacterial and eukaryotic thymidine phosphorylases and anthranilate phosphoribosyltransferases, the relationship between which has not been suspected previously. Based on the known tertiary structure of E. coli thymidine phosphorylase, structural interpretation was given to the sequence conservation in this family. The highest conservation is observed in the N‐terminal α‐helical domain, whose exact function is not known. Parts of the conserved active site of thymidine phosphorylases and anthranilate phosphoribosyltransferases were delineated. A motif in the putative phosphate‐binding site is conserved in family II and in other phosphoribosyltransferases. Our analysis suggests that certain enzymes of very similar specificity, e.g., uridine and thymidine phosphorylases, could have evolved independently. In contrast, enzymes catalyzing such different reactions as AMP hydrolysis and uridine phosphorolysis or thymidine phosphorolysis and phosphoribosyl anthranilate synthesis are likely to have evolved from common ancestors.
Self‐splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family
Authors: Gorbalenya Alexander E. 
Published: February 2009

AbstractA new family of protein domains consisting of 50–80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name “EX1H‐HX3H” was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The “His” motifs often alternate with amino‐ and carboxy‐terminal motifs of a new type of Zn‐finger‐like structure CX2, 4CX29–54[CH]X2, 3[CH]. The EX1HH‐HX3H domain in eubacterial E2‐type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH‐EX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH‐HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.
Multiple conformations of the sea anemone polypeptide anthopleurin‐A in solution
Authors: Scanlon Martin J. , Norton Raymond S. 
Published: February 2009

AbstractAnthopleurin‐A (AP‐A) is a member of a family of sea anemone‐derived polypeptides that interact with sodium channels in a voltage‐dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3‐dimensional structure of AP‐A were complicated by the fact that it was found to exist in 2 conformations in solution. Using1H‐ and13C‐NMR spectroscopy, we have now shown that this conformational heterogeneity arises fromcis–transisomerization about the Gly 40–Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts acisconformation. Furthermore, the increased sensitivity afforded by higher‐field NMR has allowed identification of additional minor conformations of AP‐A, the origin of which is presently unknown. We believe there will be many more examples of the detection by high‐field NMR of previously unobserved minor conformations of proteins in solution.
Crystallization and preliminary X‐ray crystallographic studies of UDP‐N‐acetylenolpyruvylglucosamine reductase
Authors: Benson Timothy E. , Walsh Christopher T. , Hogle James M. 
Published: February 2009

AbstractThe overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP‐N‐acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X‐ray crystallographic studies of the enzyme. MurB crystallizes in 14–20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP‐N‐acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P41212 with a = b = 49.6 Å, c = 263.2 Å, and α = β = γ = 90° at –160 °C and diffract to at least 2.5 Å. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.
Mouse liver NAD(P)H:quinone acceptor oxidoreductase: Protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression inEscherichia coli, and enzyme activity analysis
Authors: Chen Shiuan , Clarke Patrick E. , Martino Paul A. , Deng Paulis S. K. , Yeh Che‐Hung , Lee Terry D. , Prochaska H. J. , Talalay Paul 
Published: February 2009

AbstractThe amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986,J Biol Chem 261:1372–1378), that the 2 forms – the hydrophilic and hydrophobic forms – of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino‐termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification.Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transformEscherichia colistrain JM109. TheE. coli‐expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H‐menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme. The results would indicate that the regions in mouse quinone reductase that contain amino acids different from the rat and human enzymes are critical for the binding of menadione, flavones, and dicoumarol.
Structure and functions of arrestins
Authors: Palczewski Krzysztof 
Published: February 2009

AbstractTransmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these amplification reactions, receptors are precisely inactivated by mechanisms that involve protein kinases and regulatory proteins called arrestins. The structure and functions of arrestins are the focus of this review.
Subunit interface mutants of rabbit muscle aldolase form active dimers
Authors: Beernink Peter T. , Tolan Dean R. 
Published: February 2009

AbstractWe report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623–8627). The disease arises from substitution of Gly for Asp‐128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site‐directed mutagenesis is used to replace Asp‐128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild‐type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild‐type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes exist as mixtures of dimer and tetramer at 4 °C. Sedimentation at 20 °C shows that the mutant enzymes are >99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 °C compared to wild‐type enzyme. The results indicate that Asp‐128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.
The NMR solution structure of the pheromone Er‐1 from the ciliated protozoan Euplotes raikovi—NMR structure of the pheromone Er‐I
Authors: Mronga S. , Luginbühl P. , Brown L. R. , Ortenzi C. , Luporini P. , Bradshaw R. A. , WÜthrich K. 
Published: February 2009

AbstractThe 3‐dimensional structure of the pheromone Er‐1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40‐residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin—spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er‐1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 Å for the backbone atoms N, Cα, and C', and 0.93 Å for all heavy atoms of the complete polypeptide chain, residues 1–40. The molecular architecture is dominated by an up‐down‐up bundle of 3 α‐helices formed by residues 2–9, 12–19, and 24–33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er‐10, the C‐terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.
Facile transition between 310‐ and α‐helix: Structures of 8‐, 9‐, and 10‐residue peptides containing the ‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐fragment
Authors: Karle Isabella L. , Flippen‐Anderson Judith L. , Gurunath R. , Balaram P. 
Published: February 2009

AbstractA structural transition from a 310‐helix to an α‐helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc‐Aib‐Trp‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐OMe, nonapeptide (B) Boc‐Trp‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐OMe, and octapeptide (C) Boc‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐OMe, are completely helical in their respective crystals. At 0.9 Å resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 310‐helices with average torsional angles ϕ(N‐Cα) and Ψ(Cα–C') of –57°, –26° for C and –60°, –27° for B. The 10‐residue peptide (A) begins as a 310‐helix and abruptly changes to an α‐helix at carbonyl O(3), which is the acceptor for both a 4 → 1 hydrogen bond with N(6)H and a 5 → 1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average ϕ, Ψ angles in the decapeptide are –58°, –28° for residues 1–3 and –63°, –41 ° for residues 4–10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 310‐ to α‐helix transition with the amide I band appearing at 1,656–1,657 cm−1 in the 9‐ and 10‐residue peptides, whereas in shorter sequences the band is observed at 1,667 cm−1.
Optimization of the electrostatic interactions in proteins of different functional and folding type
Authors: Spassov Velin Z. , Karshikoff Andrej D. , Ladenstein Rudolf 
Published: February 2009

AbstractThe 3‐dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge‐charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge‐charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α‐helical or β‐strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge‐charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.