bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sUgar phosphotransferase system produced by substituting alanine or glutamate for the active‐site histidine 189: Implications for phosphorylation effects
Authors: Ginsburg Ann , Szczepanowski Roman H. , Ruvinov Sergei B. , Nosworthy Neil J. , Sondej Melissa , Umland Timothy C. , Peterkofsky Alan
Published: February 2009

The amino terminal domain of enzyme I (residues 1‐258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active‐site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature‐induced changes in ellipticity at 222 nm for monomeric wild‐type and mutant EIN proteins indicate two‐state unfolding. For EIN proteins in 10 mM K‐phosphate (and 100 mM KCl) at pH 7.5ΔH ≅ 140± 10 (160) kcal mol−1 and ΔCp ≅ 2.7 (3.3) kcal K−1 mol−1. Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) °C for wild‐type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho‐His 189, phospho‐His189, and H189 substitutions of EIN at pH7.5 is: His > Ala > Glu > His‐PO32− due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm ∼ 66–68 °C). This possibly arises from an ion pair interaction between the γ‐carboxyl of Glu189 and the ϵ‐amino group of Lys69 in the docking region for the histidine‐containing phosphocarrier protein HPr. However, the binding of HPr to wild‐type and active‐site mutants of EIN and EI is temperature‐independent (entropically controlled) with about the same affinity constant at pH 7.5: K'A = 3 ± 1 × 105 M−1 for EIN and ∼1.2 × 105 M−1 for EI.
Conformational behavior of ionic self‐complementary peptides
Authors: Altman Michael , Lee Peter , Rich Alexander , Zhang Shuguang
Published: February 2009

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable β‐sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N‐terminus and positively charged arginine or lysine residues located toward the C‐terminus. This arrangement of charge balances the α‐helical dipole moment (C → N), resulting in a strong tendency to form stable α‐helices as well. Therefore, these peptides can form both stable α‐helices and β‐sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable β‐sheet and α‐helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.
Prediction of amino acid sequence from structure
Authors: Raha Kaushik , Wollacott Andrew M. , Italia Michael J. , Desjarlais John R.
Published: February 2009

We have developed a method for the prediction of an amino acid sequence that is compatible with a three‐dimensional backbone structure. Using only a backbone structure of a protein as input, the algorithm is capable of designing sequences that closely resemble natural members of the protein family to which the template structure belongs. In general, the predicted sequences are shown to have multiple sequence profile scores that are dramatically higher than those of random sequences, and sometimes better than some of the natural sequences that make up the superfamily. As anticipated, highly conserved but poorly predicted residues are often those that contribute to the functional rather than structural properties of the protein. Overall, our analysis suggests that statistical profile scores of designed sequences are a novel and valuable figure of merit for assessing and improving protein design algorithms.
Tumor suppressor INK4: Refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data
Authors: Yuan Chunhua , Selby Thomas L. , Li Junan , Byeon In‐Ja L. , Tsai Ming‐Daw
Published: February 2009

Within the tumor suppressor protein INK4 (inhibitor of cyclin‐dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H‐bonds). For this purpose, the solution structure of p16INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure‐based iterative NOE assignments. The nonhelical regions showed major improvement with root‐mean‐square deviation (RMSD) improved from 1.23 to 0.68 Å for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A‐CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.
Overall rotational diffusion and internal mobility in domain II of protein G from Streptococcus determined from 15N relaxation data
Authors: Tillett Marcus L. , Blackledge Martin J. , Derrick Jeremy P. , Lian Lu‐Yun , Norwood Timothy J.
Published: February 2009

The backbone dynamics and overall tumbling of protein G have been investigated using 15N relaxation. Comparison of measured R2/R1 relaxation rate ratios with known three‐dimensional coordinates of the protein show that the rotational diffusion tensor is significantly asymmetric, exhibiting a prolate axial symmetry. Extensive Monte Carlo simulations have been used to estimate the uncertainty due to experimental error in the relaxation rates to be D∥︁/D1 = 1.68± 0.08, while the dispersion in the NMR ensemble leads to a variation of Dn/D1 = 1.65 ± 0.03. Incorporation of this tensorial description into a Lipari‐Szabo type analysis of internal motion has allowed us to accurately describe the local dynamics of the molecule. This analysis differs from an earlier study where the overall rotational diffusion was described by a spherical top. In this previous analysis, exchange parameters were fitted to many of the residues in the alpha helix. This was interpreted as reflecting a small motion of the alpha helix with respect to the beta sheet. We propose that the differential relaxation properties of this helix compared to the beta sheet are due to the near‐orthogonality of the NH vectors in the two structural motifs with respect to the unique axis of the diffusion tensor. Our analysis shows that when anisotropic rotational diffusion is taken into account NH vectors in these structural motifs appear to be equally rigid. This study underlines the importance of a correct description of the rotational diffusion tensor if internal motion is to be accurately investigated.
Influence of the C‐terminus of the glycophorin A transmembrane fragment on the dimerization process
Authors: Orzáez Mar , Pérez‐Payá Enrique , Mingarro Ismael
Published: February 2009

The monomer–dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix–helix packing in a membrane‐mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C‐terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two‐stage model for membrane protein folding and oligomerization.
The active site and substrates binding mode of malonyl‐CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site‐directed mutagenesis, and comparative modeling studies
Authors: Jung Jin‐Won , An Jae Hyung , Na Kyu Bong , Kim Yu Sam , Lee Weontae
Published: February 2009

The active sites and substrate bindings of Rhizobium trifolii molonyl‐CoA synthetase (MCS) catalyzing the malonyl‐CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site‐directed mutagenesis, and comparative modeling methods. The MCS‐bound conformation of malonyl‐CoA was determined from two‐dimensional‐transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 α‐helices, 24 β‐strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C‐terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl‐AMP. These findings are strongly supported by previous biochemical data, as well as by the site‐directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate‐forming enzymes have.
The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry
Authors: Jamin Marc , Loh Stewart N. , Baldwin Robert L. , Antalik Marian , Bolen D. Wayne , Loh Stewart N.
Published: February 2009

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 °C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 °C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.
Autopalmitoylation of tubulin
Authors: Wolff J. , Zambito Anna Maria , Britto P. Jeram , Knipling Leslie
Published: February 2009

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2–3 h at 36–37 °C under native conditions. Both α and β tubulin are labeled, and 63–73% of the label was hydroxylamine‐labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of ∼13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of α tubulin toward the β band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/ depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.
The X‐ray structure of the NAD‐dependent 5,10‐methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae
Authors: Monzingo Arthur F. , Breksa Andrew , Ernst Stephen , Appling Dean R. , Robertus Jon D.
Published: February 2009

Eucaryotes possess one or more NADP‐dependent methylene‐THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD‐dependent enzyme, yMTD. We report X‐ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 Å resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD‐binding domain that has two inserted strands compared with the NADP‐binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.
HYR, an extracellular module involved in cellular adhesion and related to the immunoglobulin‐like fold
Authors: Callebaut Isabelle , Mornon Jean‐Paul , Gilgès Delphine , Vigon Isabelle
Published: February 2009

Domains belonging to the immunoglobulin‐like fold are responsible for a wide variety of molecular recognition processes. Here we describe a new family of domains, the HYR family, which is predicted to belong to this fold, and which appears to be involved in cellular adhesion. HYR domains were identified in several eukaryotic proteins, often associated with Complement Control Protein (CCP) modules or arranged in multiple copies. Our analysis provides a sequence and structural basis for understanding the role of these domains in interaction mechanisms and leads to further characterization of heretofore undescribed repeated domains with similar folds found in several bacterial proteins involved in enzymatic activities (some chitinases) or in cell surface adhesion (streptococcal C‐alpha antigen).
Decoys ‘R’ Us: A database of incorrect conformations to improve protein structure prediction
Authors: Samudrala Ram , Levitt Michael
Published: February 2009

The development of an energy or scoring function for protein structure prediction is greatly enhanced by testing the func–tion on a set of computer–generated conformations (decoys) to determine whether it can readily distinguish native–like conforma–tions from nonnative ones. We have created “Decoys “R” Us,” a database containing many such sets of conformations, to provide a resource that allows scoring functions to be improved.
Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc–β–lactamase
Authors: Chantalat Laurent , Duée Emile , Dideberg Otto , Galleni Moreno , Frère Jean–Marie
Published: February 2009

β–Lactamases are involved in bacterial resistance. Mem–bers of the metallo–enzyme class are now found in many patho–genic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn–β–lactamase X–ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in β–lactam hydrolysis, the re–placement by Ser of the zinc–liganding residue Cys168 in the Zn–β–lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono–Zn form, but active in the di–Zn form. The structure of the mono–Zn form of the C168S mutant has been determined at 1.85 Å resolution. Ser168 occupies the same position as Cys168 in the wild–type enzyme. The protein residues mostly affected by the mutation are Asp90– Arg91 and His210. A critical factor for the activity of the mono–Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs cataly‐sis. The evolution of a large superfamily including Zn–β–lactamases suggests that they may not all share the same mechanism.
Folding of a three–stranded coiled coil
Authors: Dürr Eberhard , Bosshard Hans Rudolf
Published: February 2009

Coiled coils consist of two or more amphipathic α–helices wrapped around each other to form a superhelical structure stabi–lized at the interhelical interface by hydrophobic residues spaced in a repeating 3–4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which asso–ciation and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three–stranded coiled coil. The designed 29–residue peptide LZ16A was shown previously to be in a concen–tration–dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time–resolved fluorescence change experiments that folding of LZ16A to D and T can be described by

The following rate constants were determined (25 °C, pH 7): k1 = 7.8×104M‐1s‐1, k‐1=0.015 s‐1, k2 = 6.5×105M‐1s−1, and k‐2 = 1.1 s‐1. In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far–ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to KD = k1/2k‐1 and KT = k2/k‐2. Furthermore, pulsed hydrogen–exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are com–patible with a two–step reaction in which a subpopulation of as–sociation competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.
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

CrmA 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

Blue 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

Human 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

The 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

The 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

The 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

A 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

Colleetins 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

The 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

The 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

The 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

Transcription 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

Bile‐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

The 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.
Redox‐related conformational changes in Rhodobacter capsulatus cytochrome c2
Authors: Zhao Dezheng , Hutton Harold M. , Gooley Paul R. , Mackenzie Neil E. , Cusanovich Michael A.
Published: February 2009

WEFT‐NOESY and transfer WEFT‐NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo‐contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H‐1H and 1H‐15N two‐dimensional NMR spectroscopy. The short‐range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox‐related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo‐contact shifts and the hydrogen‐deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.
Change in dimerization mode by removal of a single unsatisfied polar residue located at the interface
Authors: Pokkuluri P. R. , Cai X. , Johnson G. , Stevens F. J. , Schiffer M.
Published: February 2009

The importance of unsatisfied hydrogen bonding potential on protein‐protein interaction was studied. Two alternate modes of dimerization (conventional and flipped form) of an immunoglobulin light chain variable domain (VL) were previously identified. In the flipped form, interface residue Gln89 would have an unsatisfied hydrogen bonding potential. Removal of this Gln should render the flipped dimer as the more favorable quaternary form. High resolution crystallographic studies of the Q89A and Q89L mutants show, as we predicted, that these proteins indeed form flipped dimers with very similar interfaces. A small cavity is present in the Q89A mutant that is reflected in the ∼100 times lower association constant than found for the Q89L mutant. The association constant of Q89A and Q89L proteins (4 × 106 M−1 and > 108 M−1) are 10‐ and 1,000‐fold higher than that of the wild‐type protein that forms conventional dimers clearly showing the energetic reasons for the flipped dimer formation.
The Effects of Internal Audit Report Disclosure on Investor Confidence and Investment Decisions
Authors: Holt Travis P. , DeZoort Todd
Published: February 2009

Current governance disclosure requirements include reports from management, the audit committee, and the external auditor. However, despite internal audit's prominence as a critical governance mechanism, external stakeholders lack information about the function. This study evaluates the extent that a descriptive Internal Audit Report (IAR) affects investor confidence and investment decisions. The results indicate that participants provided with an IAR had more confidence in financial reporting reliability and higher perceived company oversight effectiveness than participants without access to an IAR. The IAR effect on confidence in financial reporting reliability is particularly evident for high fraud risk companies. Tests for mediation reveal that the IAR effect on confidence is mediated by perceptions of oversight effectiveness. Furthermore, we find that confidence in financial reporting reliability and perceived oversight effectiveness mediate the IAR‐investment recommendation relation. Self‐insight results show that the IAR was perceived to be as useful as several currently required disclosures.
The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman‐Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies
Authors: Maserati Emanuela , Pressato Barbara , Valli Roberto , Minelli Antonella , Sainati Laura , Patitucci Francesco , Marletta Cristina , Mastronuzzi Angela , Poli Furio , Lo Curto Francesco , Locatelli Franco , Danesino Cesare , Pasquali Francesco
Published: February 2009

An investigation of 22 new patients with Shwachman‐Diamond syndrome (SDS) and the follow‐up of 14 previously reported cases showed that (i) clonal chromosome changes of chromosomes 7 and 20 were present in the bone marrow (BM) of 16 out of 36 cases, but if non‐clonal changes were taken into account, the frequency of anomalies affecting these chromosomes was 20/36: a specific SDS karyotype instability was thus confirmed; (ii) the recurrent isochromosome i(7)(q10) did not include short arm material, whereas it retained two arrays of D7Z1 alphoid sequences; (iii) the deletion del(20)(q11) involved the minimal region of deletion typical of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML); (iv) only one patient developed MDS, during the rapid expansion of a BM clone with a chromosome 7 carrying additional material on the short arms; (v) the acquisition of BM clonal chromosome anomalies was age‐related. We conclude that karyotype instability is part of the natural history of SDS through a specific mutator effect, linked to lacking SBDS protein, with consequent clonal anomalies of chromosomes 7 and 20 in BM, which may eventually promote MDS/AML with the patients’ ageing.