bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of recombinant triosephosphate isomerase from bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three‐dimensional structures points to the importance of hydrophobic interactions
Authors: Delboni Luis F. , Mande Shekhar C. , Turley Stewart , Hol WIM G.J. , Rentier‐Delrue Françoise , Mainfroid Véronique , Martial Joseph A. , Vellieux Frederique M.D. 
Published: February 2009

AbstractThe structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2‐phosphoglycolate was determined by X‐ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the “closed” conformation in either subunit. The crystallographic R‐factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 Å for 248 Cα atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual ϕ,ψ combination.A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.
Recombinant protein sequences can trigger methylation of N‐terminal amino acids in Escherichia coli
Authors: Apostol Izydor , Aitken Jackie , Levine Joe , Lippincott Julie , Davidson Jeffrey S. , Abbott‐Brown Debbie 
Published: February 2009

AbstractRecombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild‐type valine residues at all N‐termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N‐terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N‐terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N‐terminal alanine that occurred through two posttrans‐lational events: initial excision of the N‐terminal methionine, followed by methylation of alanine as the newly generated N‐terminus. No methylation was observed for variants expressed with wild‐type valine at the N‐terminus of the alpha globin. The methylation of N‐terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N‐terminal methylation has been observed previously for native E. coli proteins with similar N‐terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.
Protein recognition of ammonium cations using side‐chain aromatics: A structural variation for secondary ammonium ligands
Authors: Raine Andrew R.C. , Yang Chien‐Chih , Packman Leonard C. , White Scott A. , Mathews F. Scott , Scrutton Nigel S. 
Published: February 2009

AbstractA model for the structure of dimethylamine dehydrogenase was generated using the crystal coordinates of trimethylamine dehydrogenase. Substrate is bound in trimethylamine dehydrogenase by cation‐7r bonding, but modeling of dimethylamine dehydrogenase suggests that secondary amines are bound by a mixture of cation‐7r and conventional hydrogen bonding. In dimethylamine dehydrogenase, binding is orientationally more specific and distinct from those proteins that bind tertiary and quaternary amine groups.
Energetics of hydrogen bonding in proteins: A model compound study
Authors: Habermann Susan M. , Murphy Kenneth P. 
Published: February 2009

AbstractDifferences in the energetics of amide‐amide and amide‐hydroxyl hydrogen bonds in proteins have been explored from the effect of hydroxyl groups on the structure and dissolution energetics of a series of crystalline cyclic dipeptides. The calorimetrically determined energetics are interpreted in light of the crystal structures of the studied compounds. Our results indicate that the amide‐amide and amide‐hydroxyl hydrogen bonds both provide considerable enthalpic stability, but that the amide‐amide hydrogen bond is about twice that of the amide‐hydroxyl. Additionally, the interaction of the hydroxyl group with water is seen most readily in its contributions to entropy and heat capacity changes. Surprisingly, the hydroxyl group shows weakly hydrophobic behavior in terms of these contributions. These results can be used to understand the effects of mutations on the stability of globular proteins.
Multiple domain protein diagnostic patterns
Authors: Adams R. Mark , Das Sudeshna , Smith Temple F. 
Published: February 2009

AbstractWe have implemented an iterative algorithm for the identification of diagnostic patterns from sets of multiple‐domain proteins, where domains need not be common to all the proteins in the defining set. Our algorithm was applied to sequences gathered using a variety of methods, including BLAST, common keywords, and common E.C. numbers. In all cases, useful diagnostic patterns were obtained, possessing both high sensitivity and specificity. The patterns were found to correlate in several cases with both functional and structural domains. Patterns generated from a large number of sequence families were analyzed for probable multiple‐domain structure.
The crystal structure of TGF‐β3 and comparison to TGF‐β2: Implications for receptor binding
Authors: Mittl Peer R. E. , Priestle John P. , Cox David A. , Mcmaster Gary , Cerletti Nico , Grütter Markus G. 
Published: February 2009

AbstractTransforming growth factors β belong to a group of cytokines that control cellular proliferation and differentiation. Five isoforms are known that share approximately 75% sequence identity, but exert different biological activities. The structure of TGF‐β3 was solved by X‐ray crystallography and refined to a final R‐factor of 17.5% at 2.0 Å resolution. Comparison with the structure of TGF‐β2 (Schlunegger MP, Grütter MG, 1992, Nature 358:430‐434; Daopin S, Piez KA, Ogawa Y, Davies DR, 1992, Science 257:369‐373) reveals a virtually identical central core. Differences exist in the conformations of the N‐terminal α‐helix and in the β‐sheet loops. In TGF‐β3, the N‐terminal α‐helix has moved ≈︁ 1 Å away from the central core. This movement can be correlated with the mutation of Leu 17 to Val and Ala 47 to Pro in TGF‐β3. The β‐sheet loops rotate as a rigid body 9° around an axis that runs approximately parallel to the dimer axis. If these differences are recognized by the TGF‐β receptors, they might account for the individual cellular responses. A molecule of the precipitating agent dioxane is bound in a crystal contact, forming a hydrogen bond with Trp 32. This dioxane may occupy a carbohydrate‐binding site, because dioxane possesses some structural similarity with a carbohydrate. The dioxane is in contact with two tryptophans, which are often involved in carbohydrate recognition.
Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit
Authors: Grant Bruce D. , Tsigelny Igor , Taylor Susan S. , Adams Joseph A. 
Published: February 2009

AbstractThe active site of the cAMP‐dependent protein kinase catalytic subunit harbors a cluster of acidic residues —Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241 — that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gin (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild‐type enzyme, and displays wild‐type‐like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre‐steady‐state quench‐flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.
Cytochrome c3 from Desulfovibrio gigas: Crystal structure at 1.8 Å resolution and evidence for a specific calcium‐binding site
Authors: Matias Pedro M. , Morais José , Coelho Ricardo , Carrondo Maria Arménia , Wilson Keith , Dauter Zbigniew , Sieker Larry 
Published: February 2009

AbstractCrystals of the tetraheme cytochrome c3 from sulfate‐reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X‐ray diffraction data collected to 1.8 Å resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R‐factor of 14.9%. The three‐dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N‐terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme‐redox perturbation, and might even be involved in the specificity of recognition with its redox partner.
Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM‐3 using site‐directed mutagenesis
Authors: Govindaraj Shanthi , Poulos Thomas L. 
Published: February 2009

AbstractCytochrome P450BM‐3 is a catalytically self‐sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 54:11221‐11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin‐to‐heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six‐proline substitution, did not change significantly relative to wild‐type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin‐to‐heme electron transfer.
The protein phosphatase 2C (PP2C) superfamily: Detection of bacterial homologues
Authors: Bork Peer , Brown Nigel P. , Hegyi Hedvig , Schultz Jörg 
Published: February 2009

AbstractA thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs. These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE. Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.
Crystallization and preliminary X‐ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus
Authors: Moser Markus , Müller Rita , Jansonius Johan N. , Battchikova Natalia , Koivulehto Marianne , Korpela Timo 
Published: February 2009

AbstractRecombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X‐rays to at least 2.0 Å nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 Å, b = 93.1 Å, c = 45.6Å, α = 90.0°, β = 106.8°, γ = 90.0°. A native data set to 2.3 Å has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.
X‐ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from rhodobacter capsulatus: Implications for ion selectivity and single‐channel conductance
Authors: Przybylski Michael , Glocker Michael O. , Nestel Uwe , Schnaible Volker , Blüggel Martin , Diederichs Kay , Weckesser Jurgen , Schad Michael , Schmid Angela , Welte Wolfram , Benz Roland 
Published: February 2009

AbstractThe role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinyl‐porins determined by mass spectrometry and X‐ray crystallography. Molecular weight and peptide mapping analyses using matrix‐assisted laser desorption‐ionization mass spectrometry identified selective succinylation of three lysine‐ϵ‐amino groups (Lys‐46, Lys‐298, Lys‐300) and the N‐terminal α‐amino group. The structure of a tetra‐succinylated porin (TS‐porin) was determined to 2.4 Å and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di‐succinylated porin (MS‐porin) and a TS‐porin are localized at the inner channel surface and are solvent‐accessible: Lys‐46 is located at the channel constriction site, whereas Lys‐298, Lys‐300, and the N‐terminus are all near the periplasmic entrance of the channel. The Lys‐46 residue at the central constriction loop was modeled as succinyl‐lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS‐ and TS‐porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single‐channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure‐selective modification of charged residues as an efficient approach in the structure‐function evaluation of ion channels, and X‐ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.
The (QxW)3 domain: A flexible lectin scaffold
Authors: Hazes Bart 
Published: February 2009

AbstractLectins form a class of proteins that have evolved a specialized carbohydrate‐binding function. Based on amino acid sequence analysis, several lectin families have been described and a lectin domain, the (QxW)3 domain, was discussed recently based on 11 family members. In this paper, the (QxW)3 domain family is extended to 45 sequences, several of which have very low sequence identity with the previously known members of the family. A hidden Markov model was used to identify the most divergent members of the family. The expanded set of sequences gives us a more complete appreciation of the conserved features, and the lack thereof, in this lectin family. This, in turn, provides new insights in the structural and functional properties of the individual family members.
A novel complex of a phenolic derivative with insulin: Structural features related to the T → R transition
Authors: Smith G.David , Ciszak Ewa , Pangborn Walter 
Published: February 2009

AbstractThe structure of a symmetric T3Rf3 insulin hexamer, complexed with 4‐hydroxybenzamide, has been determined using X‐ray crystallographic techniques. Data were measured from six crystals grown in microgravity to a resolution of 1.4 Å and the structure has been refined including the contributions from hydrogen atoms. The crystals are isomorphous with T3Rf3 complexes of phenolic derivatives as well as with uncomplexed forms. Unlike the structures of complexes with phenol, m‐cresol, resorcinol, 4′‐hydroxyacetanilide, and methylparaben, which bind one phenolic derivative molecule per R‐ or Rf‐state monomer, two molecules of 4‐hydroxybenzamide are bound by each Rf‐state monomer. The presence of the second guest molecule results in an extensive hydrogen bonding network, mediated by water molecules, between the T‐ and Rf‐state trimers and adds stability to the formation of the hexamer. The only access to these second sites is through three symmetry‐related, narrow channels that originate on the surface of the T‐state trimer. Although the conformation of the backbone atoms of the monomers is nearly identical to that of other T3Rf3 hexamers, significant changes are observed in the conformations of side chains in the vicinity of the second binding site. The side chain of the T‐state A11 Cys residue, which forms a disulfide bond to A6 Cys in the same monomer, is observed in two discrete conformations; two discrete conformations are also present for the entire A8 Thr residue in the Rf‐state monomer. A procedure is also described for an alternate method of interframe scaling and merging intensity data from an image plate detector.
The NMR solution structure of the pheromone Er‐11 from the ciliated protozoan Euplotes raikovi
Authors: Luginbühl Peter , Wu Jihui , Zerbe Oliver , Ortenzi Claudio , Luporini Pierangelo , Wüthrich Kurt 
Published: February 2009

AbstractThe NMR solution structure of the pheromone Er‐11, a 39‐residue protein from the ciliated protozoan Euplotes raikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, Cα, and C of residues 2–38 was 0.30 Å. The molecular architecture is dominated by an up‐down‐up bundle of three short helices with residues 2–9, 12–19, and 22–32, which is closely similar to the previously determined structures of the homologous pheromones EM, Er‐2, and EMO. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell‐surface receptors.
Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy
Authors: Freskgård Per‐Ola , Olsen Ole H. , Persson Egon 
Published: February 2009

AbstractFactor VIIa (fVIIa) is composed of four discrete domains, a 7‐carboxyglutamic acid (Gla)‐containing domain, two epidermal growth factor (EGF)‐like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF‐like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca2+‐dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca2+‐induced changes in the far‐UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca2+‐induced ordering of α‐helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa constructed on the basis of known structures of homologous proteins. Far‐UV spectra of the Gla domain of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K‐dependent plasma proteins. The near‐UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.