bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue‐damaging activities
Authors: Watanabe Leandra , Shannon John D. , Valente Richard H. , Rucavado Alexandra , Alape‐Girón Alberto , Kamiguti Aura S. , Theakston R. David G. , Fox Jay W. , Gutiérrez José María , Arni Raghuvir K.
Published: September 2003

BaP1 is a 22.7‐kD P‐I‐type zinc‐dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue‐damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117–Cys 197, Cys 159–Cys 181, Cys 157–Cys 164). It has the consensus sequence H142E143XXH146XXGXXH152, as well as the sequence C164I165M166, which characterize the “metzincin” superfamily of metalloproteinases. The active‐site cleft separates a major subdomain (residues 1–152), comprising four α‐helices and a five‐stranded β‐sheet, from the minor subdomain, which is formed by a single α‐helix and several loops. The catalytic zinc ion is coordinated by the Nε2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active‐site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P‐I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active‐site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.
Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones
Authors: Millard Charles B. , Shnyrov Valery L. , Newstead Simon , Shin Irina , Roth Esther , Silman Israel , Weiner Lev
Published: September 2003

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is “native‐like,” and inactivation can be reversed by exposure to reduced glutathione. The allicin‐modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t1/2 ≃ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine‐N‐oxide, glycerol, and sucrose, and the divalent cations, Ca2+, Mg2+, and Mn2+ can prevent this transition of the native‐like state for >24 h at room temperature. Trimethylamine‐N‐oxide and Mg2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native‐like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.
The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites
Authors: Zhao Yonghong , Liu Deqian , Kaluarachchi Warna D. , Bellamy Henry D. , White Mark A. , Fox Robert O.
Published: September 2003

The mRNA of Escherichia coli yedU gene is induced 31‐fold upon heat shock. The 31‐kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity. We solved the crystal structure of YedU at 2.2 Å resolution. YedU monomer has an α/β/α sandwich domain and a small α/β domain. YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization. There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein. This hydrophobic patch is likely the substrate‐binding site responsible for the chaperone activity. The structure also reveals a potential protease‐like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified. YedU coordinates a metal ion using His85, His122, and Glu90. This 2‐His‐1‐carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU.
Exploring the sequence‐structure protein landscape in the glycosyltransferase family
Authors: Zhang Ziding , Kochhar Sunil , Grigorov Martin
Published: September 2003

To understand the molecular basis of glycosyltransferases' (GTFs) catalytic mechanism, extensive structural information is required. Here, fold recognition methods were employed to assign 3D protein shapes (folds) to the currently known GTF sequences, available in public databases such as GenBank and Swissprot. First, GTF sequences were retrieved and classified into clusters, based on sequence similarity only. Intracluster sequence similarity was chosen sufficiently high to ensure that the same fold is found within a given cluster. Then, a representative sequence from each cluster was selected to compose a subset of GTF sequences. The members of this reduced set were processed by three different fold recognition methods: 3D‐PSSM, FUGUE, and GeneFold. Finally, the results from different fold recognition methods were analyzed and compared to sequence‐similarity search methods (i.e., BLAST and PSI‐BLAST). It was established that the folds of about 70% of all currently known GTF sequences can be confidently assigned by fold recognition methods, a value which is higher than the fold identification rate based on sequence comparison alone (48% for BLAST and 64% for PSI‐BLAST). The identified folds were submitted to 3D clustering, and we found that most of the GTF sequences adopt the typical GTF A or GTF B folds. Our results indicate a lack of evidence that new GTF folds (i.e., folds other than GTF A and B) exist. Based on cases where fold identification was not possible, we suggest several sequences as the most promising targets for a structural genomics initiative focused on the GTF protein family.
Molecular dynamics simulation of highly charged proteins: Comparison of the particle‐particle particle‐mesh and reaction field methods for the calculation of electrostatic interactions
Authors: Gargallo Raimundo , Hünenberger Philippe H. , Avilés Francesc X. , Oliva Baldomero
Published: September 2003

Molecular dynamics (MD) simulations of the activation domain of porcine procarboxypeptidase B (ADBp) were performed to examine the effect of using the particle‐particle particle‐mesh (P3M) or the reaction field (RF) method for calculating electrostatic interactions in simulations of highly charged proteins. Several structural, thermodynamic, and dynamic observables were derived from the MD trajectories, including estimated entropies and solvation free energies and essential dynamics (ED). The P3M method leads to slightly higher atomic positional fluctuations and deviations from the crystallographic structure, along with somewhat lower values of the total energy and solvation free energy. However, the ED analysis of the system leads to nearly identical results for both simulations. Because of the strong similarity between the results, both methods appear well suited for the simulation of highly charged globular proteins in explicit solvent. However, the lower computational demand of the RF method in the present implementation represents a clear advantage over the P3M method.
Binding‐induced folding transitions in calpastatin subdomains A and C
Authors: Mucsi Zoltán , Hudecz Ferenc , Hollósi Miklós , Tompa Peter , Friedrich Peter
Published: September 2003

Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca2+‐dependent manner. The peptides are disordered in water but show a high propensity for α‐helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca2+, and is clearly seen upon binding of the peptides to the enzyme. Secondary‐structure prediction of all calpastatin sequences shows that the helix‐forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding‐induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.
On the structural and functional modularity of glycinamide ribonucleotide formyltransferases
Authors: Lee Seung‐Goo , Lutz Stefan , Benkovic Stephen J.
Published: September 2003

Glycinamide ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the glycinamide ribonucleotide substrate. Despite the low amino acid‐sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable. As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids. The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain. From a pool of several dozen sequence distinct hybrids, six in vivo‐functional fusion genes were selected, overexpressed, and purified to homogeneity. The kinetic analysis of these constructs and the comparison of their kcat and KM values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are “modular encoded” and can be exchanged by domain swapping. The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization.
Isolation and characterization of human nuclear and cytosolic multisynthetase complexes and the intracellular distribution of p43/EMAPII
Authors: Wolfe Cindy L. , Warrington J. Anthony , Davis Stanitia , Green Sherrina , Norcum Mona T.
Published: September 2003

In this study, the human multienzyme aminoacyl–tRNA synthetase “core” complex has been isolated from the nuclear and cytosolic compartments of human cells and purified to near homogeneity. It is clear from the polypeptide compositions, stoichiometries, and three‐dimensional structures that the cytosolic and nuclear particles are very similar to each other and to the particle obtained from rabbit reticulocytes. The most significant difference observed via aminoacylation activity assays and densitometric analysis of electrophoretic band patterns is a lower amount of glutaminyl–tRNA synthetase in the human particles. However, this is not enough to cause major changes in the three‐dimensional structures calculated from samples negatively stained with either uranyl acetate or methylamine vanadate. Indeed, the latter samples produce volumes that are highly similar to an initial structure previously calculated from a frozen hydrated sample of the rabbit multisynthetase complex. New structures in this study reveal that the three major structural domains have discrete subsections. This information is an important step toward determination of specific protein interactions and arrangements within the multisynthetase core complex and understanding of the particle's cellular function(s). Finally, gel filtration and immunoblot analysis demonstrate that a major biological role for the cytokine precursor p43 is as an integral part of the multisynthetase complex.
The role of protein stability, solubility, and net charge in amyloid fibril formation
Authors: Schmittschmitt Jason P. , Scholtz J. Martin
Published: September 2003

Ribonuclease Sa and two charge‐reversal variants can be converted into amyloid in vitro by the addition of 2,2,2‐triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well‐characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.
Structure and disorder in the ribonuclease S‐peptide probed by NMR residual dipolar couplings
Authors: Alexandrescu Andrei T. , Kammerer Richard A.
Published: September 2003

NMR residual dipolar couplings for the S‐peptide of ribonuclease A aligned in C8E5/n‐octanol liquid crystals are consistent with the presence of a native‐like α‐helix structure undergoing dynamic fraying. Residues 3–13, which correspond to the first α‐helix of ribonuclease A, show couplings that become more negative at low temperature and in the presence of salt, conditions which stabilize α‐helical structure in the S‐peptide. By contrast, dipolar couplings from the N and C termini of the peptide are close to zero and remain nearly invariant with changes in solution conditions. Torsion angle dynamics simulations using a gradient of dihedral restraint bounds that increase from the center to the ends of the peptide reproduce the experimentally observed sequence dependence of dipolar couplings. The magnitudes of residual dipolar couplings depend on the anisotropy of the solute. Native proteins often achieve nearly spherical shapes due to the hydrophobic effect. Embryonic partially folded structures such as the S‐peptide α‐helix have an intrinsically greater potential for anisotropy that can result in sizable residual dipolar couplings in the absence of long‐range structure.
Kinetics of the dimerization of retroviral proteases: The “fireman's grip” and dimerization
Authors: Ingr Marek , Uhlíková Tat'ána , Str̆ís̆ovský Kvido , Majerová Eva , Konvalinka Jan
Published: September 2003

All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the “fireman's grip,” which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp‐Thr(Ser)‐Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp‐Thr‐Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild‐type and mutant proteases from HIV and myeloblastosis‐associated virus harboring either Ser or Thr in their Asp‐Thr(Ser)‐Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be ∼10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.
The specificity of cross‐reactivity: Promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness
Authors: James Leo C. , Tawfik Dan S.
Published: September 2003

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross‐reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4‐dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross‐reactants are themselves bound specifically—close derivatives of these cross‐reactants show very low or no binding to SPE7. It has been suggested that cross‐reactivity is simply due to “hydrophobic stickiness”, nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross‐reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1–8. By comparing the sequences and binding patterns of SPE7 and B1–8, we address the relationship between affinity maturation, specificity, and cross‐reactivity.
Contribution of active site residues to the activity and thermal stability of ribonuclease Sa
Authors: Yakovlev Gennady I. , Mitkevich Vladimir A. , Shaw Kevin L. , Trevino Saul , Newsom Stephanie , Pace C. Nick , Makarov Alexander A.
Published: September 2003

We have used site‐specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000‐fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, kcat is reduced sixfold despite the fact that Glu 74 is over 15 Å from the active site. The pH dependence of kcat/KM is very similar for Glu74Lys and wild‐type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild‐type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.
The physical basis of microtubule structure and stability
Authors: Sept David , Baker Nathan A. , McCammon J. Andrew
Published: September 2003

Microtubules are cylindrical polymers found in every eukaryotic cell. They have a unique helical structure that has implications at both the cellular level, in terms of the functions they perform, and at the multicellular level, such as determining the left–right symmetry in plants. Through the combination of an atomically detailed model for a microtubule and large‐scale computational techniques for computing electrostatic interactions, we are able to explain the observed microtubule structure. On the basis of the lateral interactions between protofilaments, we have determined that B lattice is the most favorable configuration. Further, we find that these lateral bonds are significantly weaker than the longitudinal bonds along protofilaments. This explains observations of microtubule disassembly and may serve as another step toward understanding the basis for dynamic instability.
A de novo redesign of the WW domain
Authors: Kraemer‐Pecore Christina M. , Lecomte Juliette T.J. , Desjarlais John R.
Published: September 2003

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small β‐sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS‐WW1 and SPANS‐WW2), using only naturally occurring l‐amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS‐WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5°C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.
Profile–profile comparisons by COMPASS predict intricate homologies between protein families
Authors: Sadreyev Ruslan I. , Baker David , Grishin Nick V.
Published: September 2003

Recently we proposed a novel method of alignment–alignment comparison, COMPASS (the tool for COmparison of Multiple Protein Alignments with Assessment of Statistical Significance). Here we present several examples of the relations between PFAM protein families that were detected by COMPASS and that lead to the predictions of presently unresolved protein structures. We discuss relatively straightforward COMPASS predictions that are new and interesting to us, and that would require a substantial time and effort to justify even for a skilled PSI‐BLAST user. All of the presented COMPASS hits are independently confirmed by other methods, including the ab initio structure‐prediction method ROSETTA. The tertiary structure predictions made by ROSETTA proved to be useful for improving sequence‐derived alignments, because they are based on a reasonable folding of the polypeptide chain rather than on the information from sequence databases. The ability of COMPASS to predict new relations within the PFAM database indicates the high sensitivity of COMPASS searches and substantiates its potential value for the discovery of previously unknown similarities between protein families.
Association and dissociation kinetics of colicin E3 and immunity protein 3: Convergence of theory and experiment
Authors: Zhou Huan‐Xiang
Published: September 2003

The rapid binding of cytotoxic colicin E3 by its cognate immunity protein Im3 is essential in safeguarding the producing cell. The X‐ray structure of the E3/Im3 complex shows that the Im3 molecule interfaces with both the C‐terminal ribonuclease (RNase) domain and the N‐terminal translocation domain of E3. The association and dissociation rates of the RNase domain and Im3 show drastically different sensitivities to ionic strength, as previously rationalized for electrostatically enhanced diffusion‐limited protein–protein associations. Relative to binding to the RNase domain, binding to full‐length E3 shows a comparable association rate but a significantly lower dissociation rate. This outcome is just what was anticipated by a theory for the binding of two linked domains to a protein. The E3/Im3 system thus provides a powerful paradigm for the interplay of theory and experiment.
Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes
Authors: Osborne Michael J. , Venkitakrishnan Rani P. , Dyson H. Jane , Wright Peter E.
Published: September 2003

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. 1HN, 15N, and 13Cα resonance assignments have been made for the ternary complex with folate and oxidized NADP+ cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6‐dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of 1HN and 15N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide–ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X‐ray crystallography; there is no evidence that the open conformation observed in some X‐ray structures of E. coli DHFR are populated in solution.
β Edge strands in protein structure prediction and aggregation
Authors: Siepen Jennifer A. , Radford Sheena E. , Westhead David R.
Published: September 2003

It is well established that recognition between exposed edges of β‐sheets is an important mode of protein–protein interaction and can have pathological consequences; for instance, it has been linked to the aggregation of proteins into a fibrillar structure, which is associated with a number of predominantly neurodegenerative disorders. A number of protective mechanisms have evolved in the edge strands of β‐sheets, preventing the aggregation and insolubility of most natural β‐sheet proteins. Such mechanisms are unfavorable in the interior of a β‐sheet. The problem of distinguishing edge strands from central strands based on sequence information alone is important in predicting residues and mutations likely to be involved in aggregation, and is also a first step in predicting folding topology. Here we report support vector machine (SVM) and decision tree methods developed to classify edge strands from central strands in a representative set of protein domains. Interestingly, rules generated by the decision tree method are in close agreement with our knowledge of protein structure and are potentially useful in a number of different biological applications. When trained on strands from proteins of known structure, using structure‐based (Dictionary of Secondary Structure in Proteins) strand assignments, both methods achieved mean cross‐validated, prediction accuracies of ∼78%. These accuracies were reduced when strand assignments from secondary structure prediction were used. Further investigation of this effect revealed that it could be explained by a significant reduction in the accuracy of standard secondary structure prediction methods for edge strands, in comparison with central strands.
Detection of four oxidation sites in viral prolyl‐4‐hydroxylase by top‐down mass spectrometry
Authors: Ge Ying , Lawhorn Brian G. , ElNaggar Mariam , Sze Siu Kwan , Begley Tadhg P. , McLafferty Fred W.
Published: September 2003

Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl‐4‐hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 ± 3, and <2%, respectively; “top‐down” MS/MS of these ions show oxidation at the sites R28–V31, E95–F107, and K216 of 22%, 28%, and 34%, respectively, but with a possible (∼4%) fourth site at V125–D150. However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable ∼13% oxygen at the V125–D150 site. For the “bottom‐up” approach, detection of the ∼4% oxidation at the V125–D150 site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just ∼4% of this, but the uniqueness of its mass value (∼1–2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top‐down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V125–D150 until K216 is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl‐4‐hydroxylases the K216, H137, and D139 are conserved residues.
Staphostatins resemble lipocalins, not cystatins in fold
Authors: Rzychon Malgorzata , Filipek Renata , Sabat Artur , Kosowska Klaudia , Dubin Adam , Potempa Jan , Bochtler Matthias
Published: September 2003

Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Here, we present the 1.4 Å crystal structure of staphostatin B and show that the fold can be described as a fully closed, highly sheared eight‐stranded β‐barrel. Thus, staphostatin B is related to β‐barrel domains that are involved in the inhibition or regulation of proteases of various catalytic types and to the superfamily of lipocalins/cytosolic fatty acid binding proteins. Unexpectedly for a cysteine protease inhibitor, staphostatin B is not significantly similar to cystatins.
Dynamics of Ca2+‐saturated calmodulin D129N mutant studied by multiple molecular dynamics simulations
Authors: Likić Vladimir A. , Strehler Emanuel E. , Gooley Paul R.
Published: September 2003

Fifteen independent 1‐nsec MD simulations of fully solvated Ca2+ saturated calmodulin (CaM) mutant D129N were performed from different initial conditions to provide a sufficient statistical basis to gauge the significance of observed dynamical properties. In all MD simulations the four Ca2+ ions remained in their binding sites, and retained a single water ligand as observed in the crystal structure. The coordination of Ca2+ ions in EF‐hands I, II, and III was sevenfold. In EF‐hand IV, which was perturbed by the mutation of a highly conserved Asp129, an anomalous eightfold Ca2+ coordination was observed. The Ca2+ binding loop in EF‐hand II was observed to dynamically sample conformations related to the Ca2+‐free form. Repeated MD simulations implicate two well‐defined conformations of Ca2+ binding loop II, whereas similar effect was not observed for loops I, III, and IV. In 8 out of 15 MD simulations Ca2+ binding loop II adopted an alternative conformation in which the Thr62 〉C=O group was displaced from the Ca2+ coordination by a water molecule, resulting in the Ca2+ ion ligated by two water molecules. The alternative conformation of the Ca2+ binding loop II appears related to the “closed” state involved in conformational exchange previously detected by NMR in the N‐terminal domain fragment of CaM and the C‐terminal domain fragment of the mutant E140Q. MD simulations suggest that conformations involved in microsecond exchange exist partially preformed on the nanosecond time scale.
Finding evolutionary relations beyond superfamilies: Fold‐based superfamilies
Authors: Matsuda Keiko , Nishioka Takaaki , Kinoshita Kengo , Kawabata Takeshi , Go Nobuhiro
Published: September 2003

Superfamily classifications are based variably on similarity of sequences, global folds, local structures, or functions. We have examined the possibility of defining superfamilies purely from the viewpoint of the global fold/function relationship. For this purpose, we first classified protein domains according to the β‐sheet topology. We then introduced the concept of kinship relations among the classified β‐sheet topology by assuming that the major elementary event leading to creation of a new β‐sheet topology is either an addition or deletion of one β‐strand at the edge of an existing β‐sheet during the molecular evolution. Based on this kinship relation, a network of protein domains was constructed so that the distance between a pair of domains represents the number of evolutionary events that lead one from the other domain. We then mapped on it all known domains with a specific core chemical function (here taken, as an example, that involving ATP or its analogs). Careful analyses revealed that the domains are found distributed on the network as >20 mutually disjointed clusters. The proteins in each cluster are defined to form a fold‐based superfamily. The results indicate that >20 ATP‐binding protein superfamilies have been invented independently in the process of molecular evolution, and the conservative evolutionary diffusion of global folds and functions is the origin of the relationship between them.
Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity
Authors: Bessler Cornelius , Schmitt Jutta , Maurer Karl‐Heinz , Schmid Rolf D.
Published: September 2003

α‐Amylases, in particular, microbial α‐amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for α‐amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the α‐amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild‐type BAA and the mutants BAA S201N and BAA N297D were subjected to error‐prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5‐fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4‐ and 1.5‐fold, respectively. Another mutant (BAA 29) possesses a wild‐type‐like pH profile but possesses a 40‐fold higher activity in periplasmic extracts and a 9‐fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial α‐amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial α‐amylases.
The Value Relevance of Foreign Income: an Australian, Canadian, and British Comparison
Authors: Bodnar Gordon M. , Hwang Lee‐Seok , Weintrop Joseph
Published: September 2003

In this paper we examine the value relevance of geographical earnings disclosures for firms listed and domiciled in Australia, Canada and the United Kingdom. We find that foreign earnings in all three countries are valued differently than domestic earnings. The estimate of the association coefficient for foreign earnings changes with returns is positive in all three countries and statistically larger than the association coefficient for domestic earnings changes in Canada and the United Kingdom. Further tests show that this difference is related to relative growth opportunities of overseas operations to domestic operations. These findings are similar to results for foreign earnings association coefficients for American‐based multinationals found in Bodnar and Weintrop (1997). These results indicate that across countries the market perceives the results of foreign operations as value relevant and suggests that greater emphasis should be placed on the required disclosure of segmental data rather than on the concern that all countries prepare the segmental information using a common GAAP.
Firm‐level Disclosures and the Relative Roles of Culture and Legal Origin
Authors: Hope Ole‐Kristian
Published: September 2003

In this paper, I investigate the relative roles of legal origin and national culture in explaining firm‐level disclosure levels internationally. Using a significantly larger and more representative sample than prior research, I document, using univariate and multivariate analyses, that both legal origin and culture (as operationalized by Hofstede and Schwartz) are important in explaining firm disclosure. Neither legal origin nor culture dominates with respect to overall explanatory power for variations in disclosure levels. Consequently, it is premature to write off culture as an important factor in the financial reporting environment. Furthermore, I find that legal origin is an important conditioning variable for the role of culture. Finally, although legal origin is a key determinant of disclosure levels, I hypothesize and find that its importance decreases with the richness of a firm's information environment.
Corporate Nonfinancial Disclosure Practices and Financial Analyst Forecast Ability Across Three European Countries
Authors: Vanstraelen Ann , Zarzeski Marilyn T. , Robb Sean W. G.
Published: September 2003

This paper presents evidence that companies across three continental European countries (Belgium, Germany and the Netherlands) provide varying degrees of analyst recommended nonfinancial disclosures to the marketplace. This study is the first to examine the relationship of Jenkins Committee nonfinancial disclosure levels with the accuracy and dispersion of financial analysts' earnings forecasts. Seemingly unrelated regression tests show that larger companies and companies with a global focus voluntarily provide higher levels of both forward looking and historical nonfinancial disclosures. Additionally, higher levels of forward looking nonfinancial disclosures are associated with lower dispersion and higher accuracy in financial analysts' earnings forecasts.
Rational Speculative Bubbles: An Empirical Investigation of the London Stock Exchange
Authors: Brooks Chris , Katsaris Apostolos
Published: September 2003

In recent years, a sharp divergence of London Stock Exchange equity prices from dividends has been noted. In this paper, we examine whether this divergence can be explained by reference to the existence of a speculative bubble. Three different empirical methodologies are used: variance bounds tests, bubble specification tests, and cointegration tests based on both ex post and ex ante data. We find that, stock prices diverged significantly from their fundamental values during the late 1990's, and that this divergence has all the characteristics of a bubble.
Partial Delegation in a Model of Currency Crisis
Authors: Boinet Virginie
Published: September 2003

This paper shows that, in a fixed exchange‐rate system with an escape clause, delegating the decision on the magnitude of realignment to an inflation‐averse central banker reduces the range of realignment costs for which the policy‐maker necessarily devalues. Stressing the influence of devaluation expectations on currency crises, it is also shown that this strategy of delegation reduces the width of the multiple equilibria zone within which self‐fulfilling crises occur, thus promoting further the exchange‐rate system's stability. The higher the central banker's degree of inflation aversion, the greater is this reduction.
Utility Functions whose Parameters depend on Initial Wealth
Authors: Pedersen Christian S. , Satchell S. E.
Published: September 2003

Conventional one‐period utility functions in Economics assume that initial wealth only enters preferences through the definition of final wealth. Consequently, those utility functions most utilized (i.e., exponential and quadratic) have implausible risk characteristics. The authors characterize a new class of utility function whose risk parameters depend upon initial wealth and obtain several desirable results. In particular, investors with quadratic and exponential utility functions can have decreasing risk aversion, and risky assets in a quadratic utility multi‐asset environment do not have to be inferior as implied by the traditional framework.
Testing for Temporal Asymmetry in the Price‐Volume Relationship
Authors: Moosa Imad A. , Silvapulle Param , Silvapulle Mervyn
Published: September 2003

This paper presents some evidence for the presence of temporal asymmetry in the price‐volume relationship in the crude oil futures market. By using threshold models we show that there is bidirectional causality between volume and prices, whereas the conventional model that assumes symmetry can only detect unidirectional causality. The results also show that the price‐volume relationship is asymmetric, in the sense that negative price and volume changes have stronger effects (on each other) than positive changes. Some explanations for asymmetry in the price‐volume relationship are suggested.
Luenberger and Malmquist Productivity Indices: Theoretical Comparisons and Empirical Illustration
Authors: Boussemart Jean‐Philippe , Briec Walter , Kerstens Kristiaan , Poutineau Jean‐Christophe
Published: September 2003

This contribution establishes, from a theoretical viewpoint, the relations between the Malmquist productivity indices, that measure in either input or output orientations, and the Luenberger productivity indices, that can simultaneously contract inputs and expand outputs, but that can also measure in either input or output orientations. The main result is that a Malmquist productivity index overestimates productivity changes, since it provides productivity measures that are nearly twice those given by the Luenberger productivity index looking for simultaneous contractions of inputs and expansions of outputs. This relationship is empirically illustrated using data from 20 OECD countries over the 1974–97 period.
A Note on the Most‐Favoured‐Customer Pricing Policy
Authors: Ohnishi Kazuhiro
Published: September 2003

This paper extends the retroactive most‐favoured‐customer pricing policy examined by Cooper (1986). He showed that the policy enabled both firms in a duopoly to offer higher prices and to enjoy higher profits. This paper introduces a variable into the most‐favoured‐customer pricing policy. Then, it shows that there is an equilibrium in which the duopolists can further increase their profits.
Practitioner Review: Electroconvulsive Therapy in Adolescents
Authors: Walter Garry , Rey Joseph M. , Mitchell Philip B.
Published: September 2003

There is increasing interest regarding electroconvulsive therapy (ECT) in young persons but limited information about its optimal use. This paper reviews the indications, effectiveness, and side effects of the treatment in the teenage population and their implications for everyday clinical practice. There is particular reference to factors influencing the clinician to recommend or advise against convulsive therapy, steps in the assessment of patients, and consent issues. Practical aspects of ECT administration are also examined, including treatment locale, anaesthesia, electrode position, stimulus characteristics, EEG monitoring, and use of psychotropics during and after the ECT course.
Infantile Autism and Associated Autosomal Chromosome Abnormalities: A Register‐based Study and a Literature Survey
Authors: Lauritsen M. , Mors O. , Mortensen P. B. , Ewald H.
Published: September 2003

Infantile autism is a heterogenous disorder with unknown aetiology. Evidence from the relatively few family and twin studies suggests a genetic component. Co‐occurrence or cosegregation between infantile autism and chromosomal abnormalities may identify candidate regions, which could be tested in linkage or association studies.

The purpose of this study was to use the Danish Cytogenetic Central Register in order to detect autosomal chromosome abnormalities associated with infantile autism, and to review the literature for cases of autism associated with autosomal chromosome abnormalities to identify candidate chromosomal regions.

The register‐based study identified possible candidate regions on chromosome 7q21 and 10q21.2, which have not previously been reported.

A few interesting candidate regions, 15q11–13, 16q23, and 17p11.2 were found in the literature survey.
Childhood Conduct Problems, Hyperactivity‐impulsivity, and Inattention as Predictors of Adult Criminal Activity
Authors: Babinski Leslie M. , Hartsough Carolyn S. , Lambert Nadine M.
Published: September 2003

This study reconsiders the relationship of childhood Attention Deficit/Hyperactivity Disorder (ADHD) and childhood conduct problems with adult criminal activity by clarifying the role of the cardinal behaviors associated with the DSM‐IV ADHD subtypes (inattention and hyperactivity/impulsivity). Since their childhood (average age 9 years), 230 male and 75 female subjects have been followed prospectively and were interviewed as young adults (average age 26 at follow‐up). Early childhood behavior ratings by parents and teachers were examined to determine the role of conduct problems, hyperactivity‐impulsivity, and inattention in predicting adult criminal involvement as measured by both official arrest records and self‐report. Results show that both hyperactivity‐impulsivity and early conduct problems independently, as well as jointly, predict a greater likelihood of having an arrest record for males, but not for females. For male subjects with 10 or more self‐reported crimes, both early conduct problems and hyperactivity‐impulsivity were significant predictors, both alone and in combination. Therefore, it appears that predominantly the symptoms of hyperactivity‐impulsivity, but not inattention, contribute to the risk for criminal involvement over and above the risk associated with early conduct problems alone.
Everyday Memory Deficits in Children and Adolescents with PTSD: Performance on the Rivermead Behavioural Memory Test
Authors: Moradi Ali R. , Doost Hamid T. Neshat , Taghavi Mohammad R. , Yule William , Dalgleish Tim
Published: September 2003

Adult post‐traumatic stress disorder (PTSD) patients often report a wide range of cognitive problems in memory, concentration, attention, planning, and judgement. Evaluation of these cognitive aspects of PTSD in adults has helped to define the nature of the disorder. However, there is a paucity of such work in younger subjects. This study has employed the Rivermead Behavioural Memory Test (RBMT) to examine cognitive factors in children and adolescents with PTSD. Eighteen child and adolescent patients with PTSD and 22 control subjects completed the test. PTSD subjects showed poorer overall memory performance compared with controls. Specifically, they were worse on the prospective and orientation items of the RBMT. The results are discussed in the light of research on everyday memory in adults with PTSD.