bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro methionine oxidation of escherichia coli‐derived human stem cell factor: Effects on the molecular structure, biological activity, and dimerization
Authors: Hsu Yueh‐Rong , Narhi Linda O. , Spahr Christopher , Langley Keith E. , Lu Hsieng S. 
Published: July 2010

AbstractThe effect of oxidation of the methionine residues of Escherichia coli‐derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine‐containing peptides, using RP‐HPLC of Asp‐N endoproteinase digests. The initial oxidation rates for Met159, Met−1, Met27, Met36, and Met48 were 0.11 min−1, 0.098 min−1, 0.033 min−1 0.0063 min−1, and 0.00035 min−1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295‐nm Trp peak in the near‐UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two‐ and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.
Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences
Authors: Gryk Michael R. , Jardetzky Oleg , Klig Lisa S. , Yanofsky Charles 
Published: July 2010

AbstractTrp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA‐binding domains of both the wild‐type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA‐binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild‐type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild‐type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.
Crystallization of the Aα subunit of protein phosphatase 2A
Authors: Skidmore Michael O. , Walter Gernot , Sawaya Michael R. , Everse Stephen J. , Parkin Sean , Rupp Bernhard , Hope Håkon 
Published: July 2010

AbstractThe Aα subunit of human protein phosphatase 2A forms crystals in space group P21 with cell dimensions a = 104.0, b = 174.9, c = 168.2 Å, and β angle = 90.2°. At cryogenic temperatures, the crystals diffracted to a resolution limit of ∼ 3.0 Å. Based on the unit cell dimensions and a calculated molecular mass of 65,277 Da, the Matthews coefficient suggests eight molecules per asymmetric unit. Two native data sets were collected to a nominal resolution of 3.0 Å and merged to provide a set that is 93% complete, with Rsvm of 9.9%.
Hydrophobic folding units derived from dissimilar monomer structures and their interactions
Authors: Tsai Chung‐Jung , Nussinov Ruth 
Published: July 2010

AbstractWe have designed an automated procedure to cut a protein into compact hydrophobic folding units. The hydrophobic units are large enough to contain tertiary non‐local interactions, reflecting potential nucleation sites during protein folding. The quality of a hydrophobic folding unit is evaluated by four criteria. The first two correspond to visual characterization of a structural domain, namely, compactness and extent of isolation. We use the definition of Zehfus and Rose (Zehfus MH, Rose GD, 1986, Biochemistry 25:335–340) to calculate the compactness of a cut protein unit. The isolation of a unit is based on the solvent accessible surface area (ASA) originally buried in the interior and exposed to the solvent after cutting. The third quantity is the hydrophobicity, equivalent to the fraction of the buried non‐poiar ASA with respect to the total non‐polar ASA. The last criterion in the evaluation of a folding unit is the number of segments it includes. To conform with the rationale of obtaining hydrophobic units, which may relate to early folding events, the hydrophobic interactions are implicitly and explicitly applied in their generation and assessment. We follow Holm and Sander (Holm L, Sander C, 1994, Proteins 79:256–268) to reduce the multiple cutting‐point problem to a one‐dimensional search for all reasonable trial cuts. However, as here we focus on the hydrophobic cores, the contact matrix used to obtain the first non‐trivial eigenvector contains only hydrophobic contacts, rather than all, hydrophobic and hydrophilic, interactions. This dataset of hydrophobic folding units, derived from structurally dissimilar single chain monomers, is particularly useful for investigations of the mechanism of protein folding. For cases where there are kinetic data, the one or more hydrophobic folding units generated for a protein correlate with the two or with the three‐state folding process observed. We carry out extensive amino acid sequence order independent structural comparisons to generate a structurally non‐redundant set of hydrophobic folding units for fold recognition and for statistical purposes.
Probing the active site of α‐class rat liver glutathione S‐transferases using affinity labeling by monobromobimane
Authors: Hu Longqin , Borleske Barbara L. , Colman Roberta F. 
Published: July 2010

AbstractMonobromobimane (mBBr) is a substrate of both μ‐ and α‐class rat liver glutathione S‐transferases, with Km values of 0.63 μM and 4.9 μM for the μ‐class isozymes 3–3 and 4–4, respectively, and 26 μM for the α‐class isozymes 1–1 and 2–2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1–1 as well as μ‐class isozymes, but not of the α‐class 2–2 isozyme. Incubation of rat liver isozyme 1–1 with mBBr at pH 7.5 and 25 °C results in a time‐dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the μ‐class isozyme 3–3 and 4–4. The rate of inactivation of 1–1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S‐methyl glutathione. In contrast, 17β‐estradiol‐3,17‐disulfate (500 μM) gives a 12.5‐fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1–1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17β‐estradiol‐3,17‐disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1–1 isozyme, the other α‐class isozyme (2–2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S‐transferase isozymes.
Studies of protein‐protein interfaces: A statistical analysis of the hydrophobic effect
Authors: Tsai Chung‐Jung , Lin Shuo Liang , Wolfson Haim J. , Nussinov Ruth 
Published: July 2010

AbstractData sets of 362 structurally nonredundant protein‐protein interfaces and of 57 symmetry‐related oligomeric interfaces have been used to explore whether the hydrophobic effect that guides protein folding is also the main driving force for protein‐protein associations. The buried nonpolar surface area has been used to measure the hydrophobic effect. Our analysis indicates that, although the hydrophobic effect plays a dominant role in protein‐protein binding, it is not as strong as that observed in the interior of protein monomers. Comparison of the interiors of the monomers with those of the interfaces reveals that, in general, the hydrophobic amino acids are more frequent in the interior of the monomers than in the interior of the protein‐protein interfaces. On the other hand, a higher proportion of charged and polar residues are buried at the interfaces, suggesting that hydrogen bonds and ion pairs contribute more to the stability of protein binding than to that of protein folding. Moreover, comparison of the interior of the interfaces to protein surfaces indicates that the interfaces are poorer in polar/charged than the surfaces and are richer in hydrophobic residues. The interior of the interfaces appears to constitute a compromise between the stabilization contributed by the hydrophobic effect on the one hand and avoiding patches on the protein surfaces that are too hydrophobic on the other. Such patches would be unfavorable for the unassociated monomers in solution. We conclude that, although the types of interactions are similar between protein‐protein interfaces and single‐chain proteins overall, the contribution of the hydrophobic effect to protein‐protein associations is not as strong as to protein folding. This implies that packing patterns and interatom, or interresidue, pairwise potential functions, derived from monomers, are not ideally suited to predicting and assessing ligand associations or design. These would perform adequately only in cases where the hydrophobic effect at the binding site is substantial.
Thermodynamics of maltose binding protein unfolding
Authors: Novokhatny Valery , Ingham Kenneth 
Published: July 2010

AbstractThe maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto‐oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a ΔHcal/ΔHvH ratio in the range of 1.3–1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the TM by 8–15 °C, depending on pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The ΔHcal/ΔHvH ratio decreased to unity, indicating that the two domains melt together in a single two‐state transition. This ligand‐induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the ΔHcal/ΔH
vH ratio. Binding of maltose to MBP is characterized by very low enthalpy changes,∼‐1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity, 0.16 cal/K‐g, which is consistent with the high amount of nonpolar surface—0.72 Å2/g—that becomes accessible to solvent in the unfolded state. The high value of ΔCP determines a very steep ΔG versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5°C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.
Structures of N‐termini of helices in proteins
Authors: Doig Andrew J. , Stapley Benjamin J. , Macarthur Malcolm W. , Thornton Janet M. 
Published: July 2010

AbstractWe have surveyed 393 N‐termini of α‐helices and 156 N‐termini of 310‐helices in 85 high resolution, non‐homologous protein crystal structures for N‐cap side‐chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N‐cap sites. The following rules are generally observed for N‐capping in α‐helices: Thr and Ser N‐cap side chains adopt the gauche— rotamer, hydrogen bond to the N3 NH and have Ψ restricted to 164 ± 8°. Asp and Asn N‐cap side chains either adopt the gauche— rotamer and hydrogen bond to the N3 NH with Ψ = 172 ± 10°, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with Ψ = 107 ± 19°. With all other N‐caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N‐terminus by blocking solvation and Ψ is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N‐cap backbone C=O in more likely to form at the N‐terminus when an unfavorable N‐cap is present. In the 310‐helix Asn and Asp remain favorable N‐caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N‐cap when α‐helix formation is to be encouraged while 310‐helix formation discouraged. The strong energetic and structural preferences found for N‐caps, which differ greatly from positions within helix interiors, suggest that N‐caps should be treated explicitly in any consideration of helical structure in peptides or proteins.
Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: Secondary structure propensities are not conserved in proteins with the same fold
Authors: Ramirez‐Alvarado Marina , Serrano Luis , Blanco Francisco J. 
Published: July 2010

AbstractThe solution conformation of three peptides corresponding to the two β‐hairpins and the α‐helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non‐native locally folded structures, but no well‐defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the α‐helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non‐helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native‐like secondary structure propensities, although a minimum tendency to avoid non‐native structures and to favor native ones could be required.
LPFC: An internet library of protein family core structures
Authors: Schmidt Robert , Altman Russ B. , Gerstein Mark 
Published: July 2010

AbstractAs the number of protein molecules with known, high‐resolution structures increases, it becomes necessary to organize these structures for rapid retrieval, comparison, and analysis. The Protein Data Bank (PDB) currently contains nearly 5,000 entries and is growing exponentially. Most new structures are similar structurally to ones reported previously and can be grouped into families. As the number of members in each family increases, it becomes possible to summarize, statistically, the commonalities and differences within each family. We reported previously a method for finding the atoms in a family alignment that have low spatial variance and those that have higher spatial variance (i.e., the “core” atoms that have the same relative position in all family members and the “non‐core” atoms that do not). The core structures we compute have biological significance and provide an excellent quantitative and visual summary of a multiple structural alignment. In order to extend their utility, we have constructed a library of protein family cores, accessible over the World Wide Web at http://www‐smi.stanford.edu/projects/helix/LPFC/. This library is generated automatically with publicly available computer programs requiring only a set of multiple alignments as input. It contains quantitative analysis of the spatial variation of atoms within each protein family, the coordinates of the average core structures derived from the families, and display files (in bitmap and VRML formats). Here, we describe the resource and illustrate its applicability by comparing three multiple alignments of the globin family. These three alignments are found to be similar, but with some significant differences related to the diversity of family members and the specific method used for alignment.
The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop
Authors: Forstner Michael , Müller Alexandre , Stolz Martin , Wallimann Theo 
Published: July 2010

AbstractA histidine residue with a PKα of 7 has been inferred to act as a general acid‐base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochon‐drial isoenzyme Mib‐CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X‐ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small‐angle X‐ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild‐type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters Km or Kmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid‐base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.
Intrachain disulfide bond in the core hinge region of human IgG4
Authors: Bloom James W. , Madanat Melanie S. , Marriott David , Wong Teresa , Chan Sham‐Yuen 
Published: July 2010

AbstractIgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four‐chain structure is maintained by strong noncovalent interactions between the amino‐terminal half of pairs of heavy‐light chains and between the carboxyl‐terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy‐light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor‐a (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half‐IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571(S229P), or with an IgGl rather than IgG4 hinge region, CDP571(γ1), only trace amounts of nondisulfide bonded half‐IgG tetramers were observed. Trypsin digest reverse‐phase HPLC peptide mapping studies of CDP571 and CDP571(γ1) with on‐line electrospray ionization mass spectros‐copy supplemented with Edman sequencing identified the chemical factor preventing inter‐heavy chain disulfide bond formation between half‐IgG molecules: the two cysteines in the IgG4 and IgGl core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low‐energy conformations of 31‐33 kcal/mol and 40‐42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP pep‐tide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgGl molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgGl and thus may permit formation of a stable intrachain disulfide bond more readily.
P100, a transcriptional coactivator, is a human homologue of staphylococcal nuclease
Authors: Ponting Christopher P. 
Published: July 2010

AbstractStaphylococcus aureus nuclease (SNase) homologues, previously thought to be restricted to bacteria and archaea, are demonstrated by sequence analysis to be present also in eukary‐otes. The human cellular coactivator pi00 is shown to contain four repeats, each of which is a SNase homologue. Surprisingly, these repeats are unlikely to possess SNase‐like activities as each lacks equivalent SNase catalytic residues, yet they may mediate plOO's single‐stranded DNA‐binding function. Products of Corydalis sem‐pervirens and Saccharomyces cerevisiae open reading frames are predicted to adopt the same fold and possess similar functions as SNase. Five additional hypothetical proteins of bacterial origin are also predicted to be active SNase‐like nucleases, including one that appears to be C‐terminally truncated in a manner analogous to an engineered active SNase variant. Conservation of Asp‐19 and Asp‐83 among these homologues suggests a re‐evaluation of the roles of these residues in Ca2+‐binding and/or catalysis.