bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of cis‐biphenyl‐2,3‐dihydrodiol‐2,3‐dehydrogenase from a PCB degrader at 2.0 Å resolution
Authors: Hülsmeyer Martin , Hecht Hans‐Jürgen , Niefind Karsten , Schomburg Dietmar , Hofer Bernd , Timmis Kenneth N. , Eltis Lindsay D. 
Published: February 2009

Abstractcis‐Biphenyl‐2,3‐dihydrodiol‐2,3‐dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+‐enzyme complex was determined by molecular replacement and refined to an R‐value of 17.9% at 2.0 Å. As a member of the short‐chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two‐step reaction mechanism is proposed for cis‐dihydrodiol dehydrogenases.
Structural investigation of the A domains of human blood coagulation factor V by molecular modeling
Authors: Villoutreix Bruno O. , Dahlbäck Björn 
Published: February 2009

AbstractFactor V (FV) is a large (2,196 amino acids) nonenzymatic cofactor in the coagulation cascade with a domain Organization (A1‐A2‐B‐A3‐C1‐C2) similar to the one of factor VIII (FVIII). FV is activated to factor Va (FVa) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1‐A2) and a light chain (A3‐C1‐C2). Activated protein C (APC), together with its cofactor protein S (PS), inhibits the coagulation cascade via limited proteolysis of FVa and FVIIIa (APC cleaves FVa at residues R306, R506, and R679). The A domains of FV and FVIII share important sequence identity with the plasma copper‐binding protein ceruloplasmin (CP). The X‐ray structure of CP and theoretical models for FVIII have been recently reported. This information allowed us to build a theoretical model (994 residues) for the A domains of human FV/FVa (residues 1‐656 and 1546‐1883). Structural analysis of the FV model indicates that: (a) the three A domains are arranged in a triangular fashion as in the case of CP and the Organization of these domains should remain essentially the same before and after activation; (b) a Type II copper ion is located at the A1‐A3 interface; (c) residues R306 and R506 (cleavage sites for APC) are both solvent exposed; (d) residues 1667‐1765 within the A3 domain, expected to interact with the membrane, are essentially buried; (e) APC does not bind to FVa residues 1865‐1874. Several other features of factor V/Va, like the R506Q and A221V mutations; factor Xa (FXa) and human neutrophil elastase (HNE) cleavages; protein S, Prothrombin and FXa binding, are also investigated.
Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase
Authors: Jeffery Constance J. , Petsko Gregory A. , Ringe Dagmar , Barry Tom , Doonan Shawn 
Published: February 2009

AbstractThe crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 Å resolution in the presence of the cofactor pyridoxal‐5′‐phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R‐factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the “closed” conformation with a bound maleate inhibitor in each active site. It shares the same three‐dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.
Fold prediction by a hierarchy of sequence, threading, and modeling methods
Authors: Jaroszwski ŁUkasz , Rychlewski Leszek , Zhang Baohong , Godzik Adam 
Published: February 2009

AbstractSeveral fold recognition algorithms are compared to each other in terms of prediction accuracy and significance. It is shown that on standard benchmarks, hybrid methods, which combine scoring based on sequence‐sequence and sequence‐structure matching, surpass both sequence and threading methods in the number of accurate predictions. However, the sequence similarity contributes most to the prediction accuracy. This strongly argues that most examples of apparently nonhomologous proteins with similar folds are actually related by evolution. While disappointing from the perspective of the fundamental understanding of protein folding, this adds a new significance to fold recognition methods as a possible first step in function prediction.Despite hybrid methods being more accurate at fold prediction than either the sequence or threading methods, each of the methods is correct in some cases where others have failed. This partly reflects a different perspective on sequence/structure relationship embedded in various methods. To combine predictions from different methods, estimates of significance of predictions are made for all methods. With the help of such estimates, it is possible to develop a “jury” method, which has accuracy higher than any of the single methods. Finally, building full three‐dimensional models for all top predictions helps to eliminate possible false positives where alignments, which are optimal in the one‐dimensional sequences, lead to unsolvable sterical conflicts for the full three‐dimensional models.
In vivo acetylation identified at lysine 70 of human lens αA‐crystallin
Authors: Lin Peiping P. , Barry Richard C. , Smith David L. , Smith Jean B. 
Published: February 2009

AbstractPosttranslational modification of protein lysyl residues that change the net charge of the molecule may alter the protein conformation. Such modifications are of particular significance among lens proteins, because conformational changes are associated with the development of cataract. A previously unidentified acetylated form of αA‐crystallin has been isolated from the water‐soluble portion of human lenses. The αA‐crystallins were fractionated by anion exchange HPLC into seven peaks, each containing more than one form of αA‐crystallin. The previously reported deamidated and phosphorylated forms were identified by their molecular masses, determined by electrospray ionization mass spectrometry. In addition to these modifications, approximately 5% of αA‐crystallin had a modification that decreased the charge by one and increased the molecular mass by 42 u. This modification, identified as acetylation, was located uniquely at Lys 70. Like any modification that alters the surface charge, acetylation may affect protein conformation and intermolecular interactions, thereby altering the solubility or chaperone properties of αA‐crystallin. Acetylation of lysine 70 is potentially significant since it is located in a region that has been implicated in the chaperone activity of αA‐crystallin.
Prediction of functional residues in water channels and related proteins
Authors: Froger A. , Thomas D. , Delamarche C. , Tallur B. 
Published: February 2009

AbstractIn this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico‐chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport.
Crystal structure of an engineered cro monomer bound nonspecifically to DNA: Possible implications for nonspecific binding by the wild‐type protein
Authors: Albright Ronald A. , Matthews Brian W. , Mossing Michael C. 
Published: February 2009

AbstractThe structure has been determined at 3.0 Å resolution of a complex of engineered monomeric Cro repressor with a seven‐base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half‐operator that is recognized by each subunit of the wild‐type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence‐independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48° further out of the major groove, while the turn region of the helix‐turn‐helix remains in contact with the DNA backbone. All of the direct base‐specific interactions seen in the wild‐type Cro‐operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein‐DNA interface than for half of the wild‐type Cro‐operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence‐nonspecific interaction of the wild‐type Cro dimer with DNA. In the sequence‐specific complex, the DNA is bent, the protein dimer undergoes a large hinge‐bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.
Scan‐rate dependence in protein calorimetry: The reversible transitions of Bacillus circulans xylanase and a disulfide‐bridge mutant
Authors: Davoodi Jamshid , Wakarchuk Warren W. , Surewicz Witold K. , Carey Paul R. 
Published: February 2009

AbstractThe stabilities of Bucillus circuluns xylanase and a disulfide‐bridge‐containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild‐type protein showed a scan‐rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan‐rate dependent in the presence of urea than in the urea‐free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan‐rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706‐12712). The kinetic factors responsible for scan‐rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan‐rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide‐bridge‐containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate‐limiting step on the thermal denaturation pathway.
Engineering of betabellin‐15d: A 64 residue beta sheet protein that forms long narrow multimeric fibrils
Authors: Lim Amareth , Saderholm Matthew J. , Kroll Mathias , Yan Yibing , Perera Lalith , Erickson Bruce W. , Makhov Alexander M. , Griffith Jack D. 
Published: February 2009

AbstractThe betabellin target structure is a β‐sandwich protein consisting of two 32 residue β‐sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin‐15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p = DPrO, k = DLYS, and h = DHis) did not fold in water at pH 6.5. Air oxidation of betabellin‐15S provided betabellin‐15D, the 64 residue disulfide bridged two‐chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of β structure observed for betabellin‐15D increased with the pH and ionic strength of the solution and the betabellin‐15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin‐15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin‐15D fibrils are consistent with a single row of β‐sandwich molecules joined by multiple intersheet H‐bonds.
Transition state in the folding of α‐lactalbumin probed by the 6‐120 disulfide bond
Authors: Ikeguchi Masamichi , Kato Masao , Sugai Shintaro , Fujino Motoaki , Kuwajima Kunihiro 
Published: February 2009

AbstractThe guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of α‐lactalbumin, in which the 6‐120 disulfide bond is selectively reduced and S‐carboxymethylated, was measured and compared with that of disulfide‐intact α‐lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate‐controlled folding model and the multiple‐pathway folding model, that we had proposed previously. All of the data supported the multiple‐pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of α‐lactalbumin is not an obligatory intermediate. The cleavage of the 6‐120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6‐120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross‐link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of α‐lactalbumin is discussed.
An improved chemical approach toward the C‐terminal sequence analysis of proteins containing all natural amino acids
Authors: Hardeman Klaas , Samyn Bart , Beeumen Jozef Van , Eycken Johan Van Der 
Published: February 2009

AbstractAn improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N‐acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H‐NMR. This chemical method allows, for the first time, complete derivatization of N‐acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C‐terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C‐terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C‐terminal amino acids including proline is a major step forward in the development of a general chemical C‐terminal sequencing method that permits the C‐terminal sequence analysis of proteins of any amino acid composition.
The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA
Authors: Chen Jiwen , Smith David L. , Griep Mark A. 
Published: February 2009

AbstractDifferential chemical modification of the lysines and amino‐terminus of Escherichia coli single‐strand binding (SSB) protein was used to determine their roles in the binding of SSB to single‐stranded DNA (ssDNA). A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride. First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single‐stranded ssDNA. Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride. Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry. The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA. Acetylation rate constants were determined from time course measurements. In the absence of ssDNA, the terminal α‐amine of SSB was I0‐fold more reactive than Lys residues at positions 43, 62, 73, and 87. The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition. In the presence of ssDNA, the reactivities of the amino‐terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7‐25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA. Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer‐monomer interface.
Class‐directed structure determination: Foundation for a protein structure initiative
Authors: Terwilliger Thomas C. , Waldo Geoffrey , Peat Thomas S. , Newman Janet M. , And Kelvin Chu , Berendzen Joel 
Published: February 2009

AbstractThe recent sequencing of many complete genomes, combined with the development of methods that allow rapid structure determination for many proteins, has changed the way in which protein structure determinations can be approached. One‐by‐one determinations of individual protein structures will soon be augmented by class‐directed structure analyses in which a group of proteins is targeted and structures of representative members are determined and used to represent the entire group. Such a shift in approach would be the foundation for a broad protein structure initiative targeting classes of proteins important for biotechnology and for a fundamental understanding of protein function.
Conversion of a β‐strand to anα‐helix induced by a single‐site mutation observed in the crystal structure of fis mutant pro26Ala
Authors: Yang Wei‐Zen , Ko Tzu‐Ping , Yuan Hanna S. , Corselli Leah , Johnson Reid C. 
Published: February 2009

AbstractThe conversion from anα‐helix to a β‐strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self‐assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a β‐strand to anα‐helix by a single‐site mutation as observed in the crystal structure of Fis mutant Pro26 Ala determined at 2.0 Å resolution. Pro26 in Fis occurs at the point where a flexible extended β‐hairpin arm leaves the core structure. Thus it can be classified as a “hinge proline” located at the C‐terminal end of the β2‐strand and the N‐terminal cap of the Aα‐helix. The replacement of Pro26 to alanine extends the Aα‐helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted fromα β‐strand to anα‐helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin‐catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its β‐strand conformation in the crystal; therefore, this peptide shows a “chameleon‐like” character since it can adopt either anα‐helix or a β‐strand structure in different environments. The structure of Pro26 Ala provides an additional example where not only the protein sequence, but also non‐local interactions determine the secondary structure of proteins.
The de novo design of a rubredoxin‐like fe site
Authors: Farinas Edgardo , Regan Lynne 
Published: February 2009

AbstractA redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S‐Cys)4]−1,‐2. The design criteria for the target site included taking backbone movements into account, tetrahedral metal‐binding, and maintaining the structure and stability of the wild‐type protein. The optical absorption spectrum of the Co(II) complex of the metal‐binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal‐free and Cd(II)‐bound forms of the variant are folded correctly and are stable. The Fe(III) complex of the metal‐binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.
The structure of serine hydroxymethyltransferase as modeled by homology and validated by site‐directed mutagenesis
Authors: Pascarella Stefano , Angelaccio Sebastiana , Contestabile Roberto , Fratte Sonia Delle , Salvo Martino Di , Bossa Francesco 
Published: February 2009

AbstractWe describe a model for the three‐dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of theα‐family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site‐directed mutants. New active site features are also proposed for further experimental testing.