bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural studies by X‐ray diffraction on metal substituted desulforedoxin, a rubredoxin‐type protein
Authors: Archer M. , Carvalho A.L. , Teixeira S. , Moura I. , Moura J.J.G. , Rusnak F. , Romão M.J. 
Published: February 2009

AbstractDesulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 × 36 amino acids) protein. Each subunit contains a high‐spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal‐substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3 +, Cd2+, Hg2+, and Ni2+ salts. The In3 + and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three‐dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.
Quantum mechanical analysis of oxygenated and deoxygenated states of hemocyanin: Theoretical clues for a plausible allosteric model of oxygen binding
Authors: Fariselli Piero , Bottoni Andrea , Bernardi Ferdinando , Casadio Rita 
Published: February 2009

AbstractAbstract: In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruptus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.
Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin‐resistant boar seminal plasma O‐glycoprotein related to pB1 protein
Authors: Bezouška Karel , Sklenář Jan , Novák Petr , Halada Petr , Havlíček Vladimír , Kraus Marek , Tichá Marie , Jonáková VĚRa 
Published: February 2009

AbstractThe complete covalent structure of a novel boar DQH sperm surface protein resistant to many classical procedures of enzymatic fragmentation was determined. The relative molecular mass of the major form of this protein determined by ESI‐MS and MALDI‐MS was 13, 065.2 ±1.0 and 13, 065.1, respectively. However, additional peaks differing by 162 Da (i.e., minus hexose), 365 Da (i.e., minus hexose and N‐acetylhexosamine), 146 Da (i.e., plus deoxyhexose), and 291 Da (i.e., plus sialic acid) indicated the heterogeneity due to differences in glycosylation. The complete covalent structure of the protein was determined using automated Edman degradation, MALDI‐MS, and post‐source decay (PSD) MALDI‐MS, and shown to consist of N‐terminal O‐glycosylated peptide followed by two fibronectin type II repeats. The carbohydrates are O‐glycosidically linked to threonine 10, as confirmed by PSD MALDI‐MS of the isolated N‐terminal glycopeptide. Eight cysteine residues of the protein form four disulfide bridges, the positions of which were assigned from MALDI‐MS and Edman degradation data. We conclude that mass spectral techniques provide an indispensable tool for the detailed analysis of the covalent structure of proteins, especially those that are refractory to standard approaches of protein chemistry.
Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NAdp+ reductase: Site‐specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials
Authors: Hurley John K. , Hazzard James T. , Martínez‐Júlvez Marta , Medina Milagros , Gómez‐Moreno Carlos , Tollin Gordon 
Published: February 2009

AbstractTransient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild‐type and seven site‐specific charge‐reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein‐protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein‐protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.
The NTR module: Domains of netrins, secreted frizzled related proteins, and type I procollagen C‐proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases
Authors: Bányai László , Patthy László 
Published: February 2009

AbstractUsing homology search, structure prediction, and structural characterization methods we show that the C‐terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled‐related proteins, and (4) type I procollagen C‐proteinase enhancer proteins (PCOLCEs) are homologous with the N‐terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin‐type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin‐type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.
Dissection of the protein G B1 domain binding site for human IgG Fc fragment
Authors: Sloan David J. , Hellinga Homme W. 
Published: February 2009

AbstractThe contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine‐scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well‐defined “knobs‐into‐holes” interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar “hot spot.”
Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large ΔCp's
Authors: Sheshadri S. , Lingaraju G. M. , Varadarajan R. 
Published: February 2009

AbstractMaltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8‐anilino‐1‐naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274–329 K. At 298 K, values of ΔG°, ΔCp, and Cm were 3.1 plusmn; 0.2 kcal mol−1, 5.9 plusmn; 0.8 kcal mol−1 K−1 (15.9 cal (mol‐residue)−1 K−1), and 0.8 M, respectively, at pH3.0 and 14.5 plusmn; 0.4 kcal mol−1, 8.3 plusmn; 0.7 kcalmol−1 K−1 (22.4kcal (mol‐residue)−1 K−1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of ΔG° and ΔCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of ΔCp per mol‐residue for the molten globule is comparable to corresponding values of ΔCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of ΔCp per mol‐residue for the unfolding of the native state is among the highest currently known for any protein.
A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases
Authors: Makarova Kira S. , Aravind L. , Koonin Eugene V. 
Published: February 2009

AbstractComputer analysis using profiles generated by the PSI‐BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'.Onthe basis of the experimentally demonstrated activity of the Methano‐bacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.
Ca2+‐dependent activity of human DNase I and its hyperactive variants
Authors: Pan Clark Q. , Lazarus Robert A. 
Published: February 2009

AbstractWe have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double‐stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300‐fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild‐type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+‐dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild‐type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26‐fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate‐enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.
Predicting allosteric switches in myosins
Authors: Kirshenbaum Kent , Young Malin , Highsmith Stefan 
Published: February 2009

AbstractThe sequences of several members of the myosin family of molecular motors are evaluated using ASP (Ambivalent Structure Predictor), a new computational method. ASP predicts structurally ambivalent sequence elements by analyzing the output from a secondary structure prediction algorithm. These ambivalent sequence elements form secondary structures that are hypothesized to function as switches by undergoing conformational rearrangement. For chicken skeletal muscle myosin, 13 discrete structurally ambivalent sequence elements are identified. All 13 are located in the heavy chain motor domain. When these sequence elements are mapped into the myosin tertiary structure, they form two compact regions that connect the actin binding site to the adenosine 5′‐triphosphate (ATP) site, and the ATP site to the fulcrum site for the force‐producing bending of the motor domain. These regions, predicted by the new algorithm to undergo conformational rearrangements, include the published known and putative switches of the myosin motor domain, and they form plausible allosteric connections between the three main functional sites of myosin. The sequences of several other members of the myosin I and II families are also analyzed.
Increasing protein stability by altering long‐range coulombic interactions
Authors: Grimsley Gerald R. , Shaw Kevin L. , Fee Lanette R. , Alston Roy W. , Huyghues‐Despointes BEATRICE M.P. , Thurlkill Richard L. , Scholtz J. Martin , Pace C. Nick 
Published: February 2009

AbstractIt is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI ≈︁ 3.5 and a net charge of ≈︁ –6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 Å from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild‐type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild‐type at pH 6, where the histidine 49 side chain (pKa= 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild‐type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.
Role of the prosequence of guanylin
Authors: Schulz Axel , Marx Ute C. , Hidaka Yuji , Shimonishi Yasutsugu , Rösch Paul , Forssmann Wolf‐Georg , Adermann Knut 
Published: February 2009

AbstractGuanylin is a guanylyl cyclase (GC)‐activating peptide that is mainly secreted as the corresponding prohormone of 94 amino acid residues. In this study, we show that the originally isolated 15‐residue guanylin, representing the COOH‐terminal part of the prohormone, is released from the prohormone by cleavage of an Asp‐Pro amide bond under conditions applied during the isolation procedures. Thus, the 15‐residue guanylin is probably a non‐native, chemically induced GC‐activating peptide. This guanylin molecule contains two disulfide bonds that are absolutely necessary for receptor activation. We demonstrate that the folding of the reduced 15‐residue guanylin results almost completely in the formation of the two inactive disulfide isomers. In contrast, the reduced form of proguanylin containing the entire prosequence folds to a product with the native cysteine connectivity. Because proguanylin lacking the 31 NH2‐terminal residues of the prosequence folds only to a minor extent to guanylin with the native disulfide bonds, it is evident that this NH2‐terminal region contributes significantly to the correct disulfide‐coupled folding. Structural studies using CD and NMR spectroscopy show that native proguanylin contains a considerable amount of α‐helical and, to a lesser extent, β‐sheet structural elements. In addition, a close proximity of the NH2‐ and the COOH‐terminal regions was found by NOESY. It appears that this interaction is important for the constitution of the correct conformation and provides an explanation of the minor guanylyl cyclase activity of proguanylin by shielding the bioactive COOH‐terminal domain from the receptor.
Characterization of the hinges of the effector loop in the reaction pathway of the activation of ras‐proteins. Kinetics of binding of beryllium trifluoride to V29G and I36G mutants of Ha‐ras‐p21
Authors: Kuppens Steven , Díaz José Fernando , Engelborghs Yves 
Published: February 2009

AbstractThis work experimentally confirms the pathway of activation of Ha‐ras‐p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434–451). The process can be studied experimentally by analyzing the binding of BeF3− to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138–23143). Two mutants, V29G and I36G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.
Exchanging the active site between phytases for altering the functional properties of the enzyme
Authors: Lehmann Martin , Lopez‐Ulibarri Rual , Loch Claudia , Viarouge Céline , Wyss Markus , Van Loon Adolphus P.G.M. 
Published: February 2009

AbstractBy using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase‐1, that was 15‐26 °C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase‐1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH‐activity profile. For the new protein termed consensus phytase‐7, a major—although not complete—shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 °C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase‐7 still was >7 °C more thermostable than all wild‐type ascomycete phytases known to date. Thus, combination of the consensus approach with the selection of a “preferred” active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members.
Two‐state vs. multistate protein unfolding studied by optical melting and hydrogen exchange
Authors: Mayne Leland , Englander S. Walter 
Published: February 2009

AbstractA direct conflict between the stabilization free energy parameters of cytochrome c determined by optical methods and by hydrogen exchange (HX) is quantitatively explained when the partially folded intermediates seen by HX are taken into account. The results support the previous HX measurements of intermediate populations, show how intermediates can elude the standard melting analysis, and illustrate how they confuse the analysis when they are significantly populated within the melting transition region.