bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of the 2[4Fe‐4S] ferredoxin from Chromatium vinosum: Evolutionary and mechanistic inferences for [3/4Fe‐4S] ferredoxins
Authors: Moulis Jean‐Marc , Sieker Larry C. , Wilson Keith S. , Dauter Zbigniew 
Published: February 2009

AbstractThe crystal structure of the 2[4Fe‐4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 Å resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N‐terminal domain coordinates the two [4Fe‐4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six‐residue insertion between two ligands of one cluster forming a two‐turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH—S H‐bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5‐turn α‐helix at the C‐terminus that covers one side of the same cluster and is linked to the cluster‐binding domain by a six‐residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe‐4S] ferredoxins from a common ancestral cluster‐binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.
Peptide rescue of an N‐terminal truncation of the stoffel fragment of Taq DNA polymerase
Authors: Vainshtein Inna , Malcolm Bruce A. , Atrazhev Alexey , Elliott John F. , Eom Soo Hyun , Wishart David S. 
Published: February 2009

AbstractDeletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5′ to 3′ exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N‐terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the StofΔ12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the StofΔ47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33‐amino acid synthetic peptide, based on this critical region (i.e., residues 303–335 inclusive), was able to restore 85% of the StofΔ12 activity when added back to the truncated StofΔ47 protein as well as return the temperature optimum to that of the StofΔ12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302–336 of the enzyme form a three‐stranded β‐sheet structure that interacts with the remainder of the protein. CD analysis of the 33‐amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% β‐sheet content. These data suggest that this 33‐amino acid peptide constitutes a stable β‐sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well‐documented complementation of Ribonuclease S protein by the 15‐residue, α‐helical, S peptide.
Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands
Authors: Uversky Vladimir N. , Protasova Natalya YU. , Rogov Vladimir V. , Vassilenko Konstantin S. , Gudkov Anatoly T. , Kutyshenko Viktor P. 
Published: February 2009

AbstractIt is obvious that functional activity of a protein molecule is closely related to its structure. On the other hand, the understanding of structure‐function relationship still remains one of the intriguing problems of molecular biology. There is widespread belief that mutagenesis presents a real way to solve this problem. Following this assumption, we have investigated the effect of circular permutation in dihydrofolate reductase from E. coli on protein structure and functioning. It has been shown that in the absence of ligands two circularly permuted variants of dihydrofolate reductase possess all the properties of the molten globule state. However, after addition of ligands they gain the native‐like structural properties and specific activity. This means that the in vitro folding of permuted dihydrofolate reductase is terminated at the stage of the molten globule formation. Interaction of permuted protein with ligands leads to the structural adjustment and formation of active protein molecules.
Two partially unfolded states oftorpedo californicaacetylcholinesterase
Authors: Kreimer David I. , Shin Irina , Silman Israel , Shnyrov Valery L. , Villar Enrique , Weiner Lev 
Published: February 2009

AbstractChemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231in each subunit of a disulfide‐linked dimer ofTorpedo californicaacetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1–2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physicochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long‐lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thiol‐disulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.
Homology modeling of human methylmalonyl‐CoA mutase: A structural basis for point mutations causing methylmalonic aciduria
Authors: Thomäu Nicolas H. , Leadlay Peter F. 
Published: February 2009

AbstractPoint mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)‐dependent methylmalonyl‐CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic α‐subunit of the heterodimeric methylmalonyl‐CoA mutase from the bacterium Propionibacte‐rium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three‐dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X‐ray crystal structure of the P. shermanii methylmalonyl‐CoA mutase. Eight mutations are found in the C‐terminal B12‐binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin‐like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent‐exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N‐terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl‐CoA esters gain access to the active site (W105R). The extreme N‐terminus of methylmalonyl‐CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.
Crystallization and preliminary X‐ray diffraction studies of the Lb proteinase from foot‐and‐mouth disease virus
Authors: Guarnéa Alba , Verdaguer Núaria , Fita Ignasi , Kirchweger Regina , Liebig Hans‐Dieter , Blaas Dieter , Skern Tim 
Published: February 2009

AbstractDifferent crystal forms of the C23A mutant from the leader proteinase of foot‐and‐mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2and PEG 6000 as precipitants. Well‐developed crystals, with cubic morphology growing to approximately 1.0 mm3in size, presented a large unit cell parameter of 274.5 Å and diffracted to, at most, 5 Å resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 × 0.3 × 0.7 mm3, diffracted to approximately 4.0 Å resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 × 0.3 × 1.2 mm3diffracted beyond 3.5 Å resolution and were stable to X‐ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group 1222 (or I212121) and unit cell dimensionsa= 65.9 Å,b= 104.3 Å, andc= 124.0 Å, and appear well suited for high‐resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.
Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin
Authors: Lombardo A. , Wang Y. , Ni C.‐Z. , Dai X. , Kodandapani R. , Chiang S. , White C.A. , Pio F. , Ruoslahti E. , Ely K.R. , Xuong N‐H. , Hamlin R.C. , Dickinson C.D. 
Published: February 2009

AbstractFibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell‐binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell‐binding domain of human fibronectin that participate in a newly characterized fibronectin‐fibronectin interaction with FNIII, were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10–11 grew in the space group C2 with a = 117.1 Å, b = 38.6 Å, c = 80.6 Å, β = 97.2°, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 Å resolution. Fragment FNIII8–11 and a shorter fragment, FNIII8–10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 Å. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell‐binding domain of fibronectin is discussed.
A fragment of staphylococcal nuclease with an OB‐fold structure shows hydrogen‐exchange protection factors in the range reported for “molten globules”
Authors: Alexandrescu Andrei T. , Dames Sonja A. , Wiltscheck Ronald 
Published: February 2009

AbstractHydrogen‐exchange rates for an OB‐fold subdomain fragment of staphylococcal nuclease have been measured at pH 4.7 and 4 °C, conditions close to the minimum of acid/base catalyzed exchange. The strongest protection from solvent exchange is observed for residues from a five‐stranded β‐barrel in the NMR structure of the protein. Protection factors, calculated from the experimental hydrogen‐exchange rates, range between 1 and 190. Similarly small protection factors have in many cases been attributed to “molten globule” conformations that are supposed to lack a specific tertiary structure. The present results suggest that marginal protection from solvent exchange does not exclude well‐defined structure.
High‐resolution structures of the bifunctional enzyme and transcriptional coactivator DCoH and its complex with a product analogue
Authors: Cronk Jeff D. , Endrizzi James A. , Alber Tom 
Published: February 2009

AbstractDCoH, the dimerization cofactor of hepatocyte nuclear factor 1 (HNF‐1), functions as both a transcriptional coactivator and a pterin dehydratase. To probe the relationship between these two functions, the X‐ray crystal structures of the free enzyme and its complex with the product analogue 7,8‐dihydrobiopterin were refined at 2.3 Å resolution. The ligand binds at four sites per tetrameric enzyme, with little apparent conformational change in the protein. Each active‐site cleft is located in a subunit interface, adjacent to a prominent saddle motif that has structural similarities to the TATA binding protein. The pterin binds within an arch of aromatic residues that extends across one dimer interface. The bound ligand makes contacts to three conserved histidines, and this arrangement restricts proposals for the enzymatic mechanism of dehydration. The dihedral symmetry of DCoH suggests that binding to the dimerization domain of HNF‐1 likely involves the superposition of two‐fold rotation axes of the two proteins.
Surface point mutations that significantly alter the structure and stability of a protein's denatured state
Authors: Smith Catherine K. , Bu Zimei , Engelman Donald M. , Regan Lynne , Anderson Karen S. , Sturtevant Julian M. 
Published: February 2009

AbstractSignificantly different m values (1.9–2.7 kcal mol−1 M−1) were observed for point mutations at a single, solvent‐exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m‐value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m−) m‐value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 s−1 for the m+ mutant and 952 s−1 for the m− mutant) and the rates of unfolding (11 s−1 for m+ mutant and 3 s−1 for the m− mutant) were significantly different, as determined by stopped‐flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (α++ = 0.3). Small‐angle X‐ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd≈︁23 Å); whereas, the Rgd of the m− mutant increased from approximately 17 Å to 23 Å with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl‐induced denatured state of these two proteins.
Bending of the calmodulin central helix: A theoretical study
Authors: Spoel David Van Der , Groot Bert L. De , Hayward Steven , Berendsen Herman J. C. , Vogel Hans J. 
Published: February 2009

AbstractThe crystal structure of calcium‐calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is α‐helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicated the presence of a flexible backbone between residues Lys 77 and Asp 80. This flexibility is related directly to the function of the protein because it enables the N‐ and C‐terminal domains of the protein to move toward each other and bind to the CaM‐binding domain of a target protein. We have investigated the flexibility of the CaM central helix by a variety of computational techniques: molecular dynamics (MD) simulations, normal mode analysis (NMA), and essential dynamics (ED) analysis. Our MD results reproduce the experimentally determined location of the bend in a simulation of only the CaM central helix, indicating that the bending point is an intrinsic property of the α‐helix, for which the remainder of the protein is not important. Interestingly, the modes found by the ED analysis of the MD trajectory are very similar to the lowest frequency modes from the NM analysis and to modes found by an ED analysis of different structures in a set of NMR structures. Electrostatic interactions involving residues Arg 74 and Asp 80 seem to be important for these bending motions and unfolding, which is in line with pH‐dependent NMR and CD studies.
In vitro renaturation of bovine β‐lactoglobulin A leads to a biologically active but incompletely refolded state
Authors: Subramaniam Vinod , Steel Duncan G. , Gafni Ari 
Published: February 2009

AbstractWhen bovine β‐lactoglobulin (β‐LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room‐temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was ∽10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (∽20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133–1136), we found no changes in either the lability or phosphorescence decays of β‐LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414–22419), using conformation‐specific monoclonal antibodies to recognize native‐like structure, that long‐term changes occur in the protein conformation, compared with the native structure, on refolding.
Crystallization and preliminary X‐ray diffraction studies of α‐amylase from the antarctic psychrophile Alteromonas haloplanctis A23
Authors: Aghajari Nushin , Haser Richard , Feller Georges , Gerday Charles 
Published: February 2009

AbstractA cold‐active α‐amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 °C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold‐adapted enzyme have been initiated because a three‐dimensional structure of a mesophilic counterpart, pig pancreatic α‐amylase, already exists. α‐Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic α‐amylase, has been crystallized and data to 1.85 Å have been collected. The space group is found to be C2221 with a = 71.40 Å, b = 138.88 Å, and c = 115.66 Å. Until now, a three‐dimensional structure of a psychrophilic enzyme is lacking.
Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks
Authors: Blom Nikolaj , Hansen Jan , Brunak Søren , Blaas Dieter 
Published: February 2009

AbstractPicornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2Apro and 3Cpro. The cleavage of VP0 (giving rise to VP2 and VP4), which is carried out by a so‐far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases.A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e‐mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dk/services/NetPicoRNA/.
Relationship between protein structure and geometrical constraints
Authors: Lund Ole , Hansen Jan , Brunak Søren , Bohr Jakob 
Published: February 2009

AbstractWe evaluate to what extent the structure of proteins can be deduced from incomplete knowledge of disulfide bridges, surface assignments, secondary structure assignments, and additional distance constraints. A cost function taking such constraints into account was used to obtain protein structures using a simple minimization algorithm. For small proteins, the approximate structure could be obtained using one additional distance constraint for each amino acid in the protein. We also studied the effect of using predicted secondary structure and surface assignments. The constraints used in this approach typically may be obtained from low‐resolution experimental data. When using a cost function based on distances, half of the resulting structures will be mirrored, because the resulting structure and its mirror image will have the same cost. The secondary structure assignments were therefore divided into chirality constraints and distance constraints. Here we report that the problem of mirrored structures, in some cases, can be solved by using a chirality term in the cost function.