bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition
Authors: Simonovic Miljan , Gettins PETER G. W. , Volz Karl 
Published: February 2009

AbstractCrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS‐stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop‐cleaved crmA to 2.9 Å resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for β sheet A, whose sequence and length are unique to crmA.
Blue copper proteins: A comparative analysis of their molecular interaction properties
Authors: De Rienzo F. , Gabdoulline R.R. , Menziani M. C. , Wade R.C. 
Published: February 2009

AbstractBlue copper proteins are type‐I copper‐containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semi quantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally‐determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (1) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.
Five recombinant fragments of human serum albumin—tools for the characterization of the warfarin binding site
Authors: Dockal Michael , Chang Martina , Carter Daniel C. , Rüker Florian 
Published: February 2009

AbstractHuman serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N‐terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well‐preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA‐DOM I‐II and HSA, followed by HSA‐DOM IB‐II, HSA‐DOM II, and HSA‐DOM I‐IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand‐alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.
Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization
Authors: Pertinhez Thelma A. , Hamada Daizo , Smith Lorna J. , Chiti Fabrizio , Taddei Niccoló , Stefani Massimo , Dobson Christopher M. 
Published: February 2009

AbstractThe folding kinetics of human common‐type acylphosphatase (cAcP) from its urea‐ and TFE‐denatured states have been determined by stopped‐flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53–Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly α‐helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53–Gly69 of cAcP indicate that only the native‐like trans isomer of the Gly–Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an ∼1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53–Pro54 peptide bond is stabilized in TFE by the formation of a nonnative‐like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.
Function of a conserved sequence motif in biotin holoenzyme synthetases
Authors: Kwon Keehwan , Beckett Dorothy 
Published: February 2009

AbstractThe biotin holoenzyme synthetases (BHS) are essential enzymes in all organisms that catalyze post‐translational linkage of biotin to biotin‐dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine‐rich sequence, GRGRXG, constitutes one of the homologous regions in these enzymes and, based on its similarity to sequences found in a number of mononucleotide binding enzymes, has been proposed to function in ATP binding in the BHSs. In the Escherichia coli enzyme, the only member of the family for which a three‐dimensional structure has been determined, the conserved sequence is found in a partially disordered surface loop. Mutations in the sequence have previously been isolated and characterized in vivo. In this work these single‐site mutants, G115S, R118G, and R119W, of the E. coli BHS have been purified and biochemically characterized with respect to binding of small molecule substrates and the intermediate in the biotinylation reaction. Results of this characterization indicate that, rather than functioning in ATP binding, this glycine‐rich sequence is required for binding the substrate biotin and the intermediate in the biotinylation reaction, biotinyl‐5′‐AMP These results are of general significance for understanding structure‐function relationships in biotin holoenzyme synthetases.
Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C‐lobe of ovotransferrin
Authors: Muralidhara B. K. , Hirose Masaaki 
Published: February 2009

AbstractThe interdomain disulfide bond present in the C‐lobe of all the transferrins was postulated to restrict the domain movement resulting in the slow rate of iron uptake and release. In the present study, the conformational stability and iron binding properties of a derivative of the isolated C‐lobe of ovotransferrin in which the interdomain disulfide bond, Cys478‐Cys671 was selectively reduced and alkylated with iodoacetamide were compared with the disulfide intact form at the endosomal pH of 5.6. Pyrophosphate and chloride mediated iron release kinetics showed no difference between the disulfide‐intact and disulfide‐reduced/alkylated forms; the two protein forms yielded similar observed rate constants showing an apparent hyperbolic dependency for anion concentrations. The conformational stability evaluated by unfolding and refolding experiments was greater for the disulfide‐intact form than for the disulfide‐reduced/alkylated form: the ΔGD values at 30°C obtained by using urea were 9.0 ± 0.8 and 6.0 ± 0.4 kJ/mol for the former and latter protein forms, respectively, and the corresponding values obtained by using guanidine hydrochloride were 6.2 ± 0.9 and 4.3 ± 0.5 kJ/mol. The dissociation constant of iron (kd) was almost the same for the two protein forms, and it varied only subtly with urea concentrations but increased markedly with GdnHCl concentrations. The nonidentical values of ΔGD and kd for urea and GdnHCl can be attributed to the ionic nature of the later denaturant, in which chloride anion may influence the structure and iron uptake‐release properties of the ovotransferrin C‐lobe. Taken together, we conclude that the interdomain disulfide bond has no effect on the iron uptake and release function but significantly decreases the conformational stability in the C‐lobe.
A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases
Authors: Ayala Youhna M. , Cera Enrico Di 
Published: February 2009

AbstractA simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k‐1), acylation (k2), and deacylation (k3) in the ‐1 in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis‐Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild‐type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+ binding and site‐directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.
Collectin structure: A review
Authors: Håkansson Kjell , Reid Kenneth B. M. 
Published: February 2009

AbstractColleetins are animal calcium dependent lectins that target the carbohydrate structures on invading pathogens, resulting in the agglutination and enhanced clearance of the microorganism. These proteins form trimers that may assemble into larger oligomers. Each polypeptide chain consists of four regions: a relatively short N‐terminal region, a collagen like region, an α‐helical coiled‐coil, and the lectin domain. Only primary structure data are available for the N‐terminal region, while the most important features of the collagen‐like region can be derived from its homology with collagen. The structures of the α‐helical coiled‐coil and the lectin domain are known from crystallographic studies of mannan binding protein (MBP) and lung surfactant protein D (SP‐D). Carbohydrate binding has been structurally characterized in several complexes between MBP and carbohydrate; all indicate that the major interaction between carbohydrate and collectin is the binding of two adjacent carbohydrate hydroxyl group to a collectin calcium ion. In addition, these hydroxyl groups hydrogen bond to some of the calcium amino acid ligands. While each collectin trimer contains three such carbohydrate binding sites, deviation from the overall threefold symmetry has been demonstrated for SP‐D, which may influence its binding properties. The protein surface between the three binding sites is positively charged in both MBP and SP‐D.
Methionine oxidation within the cerebroside‐sulfate activator protein (CSAct or Saposin B)
Authors: Whitelegge Julian P. , Penn Brandon , To Trang , Johnson Jeff , Waring Alan , Sherman Mark , Stevens Richard L. , Fluharty Claire B. , Faull Kym F. , Fluharty Arvan L. 
Published: February 2009

AbstractThe cerebroside‐sulfate activator protein (CSAct or Saposin B) is a small water‐soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide‐sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed‐phase high‐performance liquid chromatography (RP‐HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithio‐threitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. Highresolution RP‐HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface‐exposed methionyl side chains, in general accordance with the existing molecular model. A mass‐spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine‐sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30‐50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.
A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein
Authors: Roesler Keith R. , Rao A. Gururaj 
Published: February 2009

AbstractThe potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor‐2 (CI‐2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three‐dimensional structure of CI‐2 and an alignment of homologous sequences. The initial engineered protein folded into a wild‐type‐like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild‐type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild‐type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild‐type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI‐2 with minimal disruption of stability and structural integrity.
Protein engineering as a strategy to avoid formation of amyloid fibrils
Authors: Villegas Virtudes , Zurdo Jesús , Filimonov Vladimir V. , Avilés Francesc X. , Dobson Christopher M. , Serrano Luis 
Published: February 2009

AbstractThe activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well‐defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.
Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription
Authors: Chen Hung‐Ta , Legault Pascale , Glushka John , Omichinski James G. , Scott Robert A. 
Published: February 2009

AbstractTranscription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA‐binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc‐binding motif near the N‐terminus that is directly involved in the interaction with RNA pol II/ TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N‐terminal residues 2–59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and “rubredoxin” turns, three antiparallel β–strands, and disordered loops. The structure is similar to the reported zinc‐ribbon motifs in several transcription‐related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc‐ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the β–sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc‐binding site may increase the stability of the β–sheet.
Crystal structure of the catalytic domain of human bile salt activated lipase
Authors: Terzyan Simon , Wang Chi‐Sun , Downs Deborah , Hunter Bret , Zhang Xuejun C. 
Published: February 2009

AbstractBile‐salt activated lipase (BAL) is a páncreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1‐538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 Å resolution. The crystal form belongs to space group P212121 with one monomer per asymmetric unit, and the protein shows an α/β hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115‐125 and 270‐285).
Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives
Authors: Ni Da Qun , Shaffer Jennifer , Adams Joseph A. 
Published: February 2009

AbstractThe binding of the methylanthraniloyl derivatives of ATP (mant‐ATP), ADP (mant‐ADP), 2′deoxyATP (mant‐2′deoxyATP), and 3′deoxyATP (mant‐3′deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant‐ADP is identical to that for ADP, while that for mant‐ATP is approximately threefold higher than that for ATP. The dissociation constant for mant‐3′deoxyATP is approximately fivefold higher than that for 3′deoxyATP while derivatization of 2′deoxyATP does not affect affinity. The time‐dependent binding of mant‐ATP, mant‐2′deoxyATP, and mant‐ADP, measured using stopped‐flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant‐ATP are equivalent substrates, as judged by reversed‐phase chromatography, although the rate of phosphorylation is approximately 20‐fold lower than the natural nucleotide. The kinetic data are consistent with a three‐step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three‐dimensional models for the enzyme.