bluuebunny/crossref_metadata_2025 huggingface dataset:
Couples Coping with Stress
Authors: OBrien Tess Byrd , DeLongis Anita , Pomaki Georgia , Puterman Eli , Zwicker Amy
Published: February 2009

The primary objective of the study was to increase understanding of interpersonal dimensions of stress and coping within married couples. Our sample included 82 couples living in a stepfamily context. Data were collected using structured telephone interviews and twice-daily questionnaires for a period of 1 week. Using matched-pair hierarchical linear modeling analysis, the study examined how stress and coping processes unfold over the course of a given day and across days within couples. First, we investigated antecedents of empathic responding, a form of relationship-focused coping. Second, we examined the role of empathic responding in within-couple variations in marital tension across days. We found that when greater personal significance was attached to family stressors, husbands and wives tended to increase their use of empathic responding. Also considered were the contextual effects of marital adjustment on how family stressors are experienced and managed by couples. The results indicate a link between marital adjustment and the use of empathic responding for both husbands and wives within couples. As well, the study suggests that marital adjustment plays an important role in determining whether the negative effects of stress will persist across days. Higher use of empathic responding was found to be associated with lower levels of next-day marital tension. When relational outcomes are considered, empathic responding may represent an adaptive way of coping with everyday stress. Our findings indicate that examination of relationship-focused coping may add to the theoretical and explanatory power of current models of stress and coping.
Resilience
Authors: Leipold Bernhard , Greve Werner
Published: February 2009

Traditionally resilience is viewed as an important way of coping: Through resilience, an individual recovers from or avoids negative outcomes from burdensome conditions. In this paper, we argue that individual stability under significant adverse conditions (i.e., resilience) results, to a large degree, from coping processes (e.g., assimilation and accommodation) influenced by personal and situational conditions. Moreover, we propose that resilience, viewed as a stabilizing constellation, should be considered an important part of the conceptual bridge between coping and development. Resilience, however, requires a definition of successful development. Here, we argue that the potential and possibility for further development may be used as a common denominator of current proposals. The aim is to outline an integrative model of coping, resilience, and development as a theoretical access to successful aging. Some empirical results illustrate the developmental conditions for assimilative and accommodative processes.
Self-Efficacy and Health-Related Outcomes of Collective Trauma
Authors: Luszczynska Aleksandra , Benight Charles C. , Cieslak Roman
Published: February 2009

The objective of our study was to systematically review research evidence for relationships between self-efficacy beliefs and psychological as well as somatic outcomes of collective traumatic events. Twenty-seven studies enrolling adult and adolescent survivors of acute, escalating, and chronic collective trauma with a total of N = 8011 participants were reviewed. Cross-sectional studies suggest medium to large effects of self-efficacy on general distress, severity and frequency of PTSD (posttraumatic stress disorder) symptoms (weighted r values range from –.36 to –.77), whereas longitudinal studies indicate large effects on general distress and PTSD symptom severity (weighted r values range: –.55 to –.62). Self-efficacy was also related to better somatic health (self-reported symptoms, i.e., less pain, fatigue, or disability). Studies addressing the relationship between self-efficacy and substance abuse after collective trauma revealed a more complex picture. Some types of pretreatment self-efficacy (e.g., self-efficacy for coping with urges) or changes in efficacy beliefs may predict less substance use or relapses. Studies testing the mediating role of cognitive or social variables in the relationship among efficacy beliefs and health outcomes indicated rather direct, unmediated effects of beliefs about ability to deal with adversities on posttraumatic adaptation. Men may benefit more from stronger efficacy beliefs. In terms of reciprocity between self-efficacy and health, evidence from longitudinal studies suggested that self-efficacy determines health-related outcomes, but changes in diagnosis do not predict changes in self-efficacy. Although a lack of experimental studies limits the conclusions, the results indicated that self-efficacy is a powerful predictor of posttraumatic recovery among collective trauma survivors.
Stress and Coping with Womens Health Issues
Authors: Benyamini Yael
Published: February 2009

The goals of this review are: (1) to present the Leventhal common-sense model (CSM) of self-regulation of stress and coping with health threats as well as new directions and questions arising from this model, and (2) to apply this theoretical perspective to womens health issues in order to highlight concerns that are unique to women. Examples from research on womens health are reviewed to show: (a) how women apply decision rules to the internal and external information available to them in order to interpret their symptoms, in an attempt to reach a coherent representation of the health threat; (b) how these representations are related to womens choices of coping strategies and why they are often unrelated to actual coping; and (c) how women appraise their situation and in what ways this internal appraisal could differ from objective outcomes. Viewing coping with womens health issues through the lens of the CSM highlights the ways in which biological and social gender differences in the experience of illness and the interactions between them affect each stage of the self-regulation process (i.e., womens experience of the stressor and the representations they form, how these representations guide coping, and womens appraisal of their situation). This review helps identify principles and general conclusions derived from the CSM and generalizes them across various threats to womens health, which should be taken into account when planning theoretically-based interventions to support women coping with health threats, as well as questions that should be investigated in future research.
Commentary on the Special Section Theory-Based Approaches to Stress and Coping
Authors: Folkman Susan
Published: February 2009

The six articles that comprise the special section on stress and coping each illuminate a different facet of the stress process. The issues addressed in these studies of dyadic coping, collective trauma, gender issues, and protective coping provide new information and, as is inevitable, raise new questions. Several studies also demonstrate some of the challenges inherent in the study of a dynamic, multilevel, recursive system that is anchored in the appraisal process of an individual in a given social context. This commentary reviews the issues and challenges raised in these studies, and suggests next steps for research in this field.
Sexualized Innocence
Authors: Machia Marty , Lamb Sharon
Published: February 2009

The present study tested the hypothesis that viewing images of women posed as sexy young girls can lead to greater acceptance of child sexual abuse myths. Sixty-five participants were randomly assigned to three groups: the control group who viewed “Nature” ads, the “Sexy Adult” group who viewed ads of adult women, and the “Sexy Child” group who viewed advertisements of women posed as sexy young girls. It was predicted that participants who viewed advertisements of women posed as sexy young girls would score higher on the Child Sexual Abuse Myth Scale (CSAM; Collings, 1997 ) than participants in the two other groups. The hypothesis was not supported; however participants who saw ads of any women scored significantly higher on the CSAM Scale than those who viewed nature ads.
Cyberbullying: Who Are the Victims?
Authors: Katzer Catarina , Fetchenhauer Detlef , Belschak Frank
Published: February 2009

Bullying is not a phenomenon exclusive to the school environment. Pupils also become victims of verbal aggression (teasing, threats, insults, or harassment) in the context of internet chatrooms. The present study addresses the following questions: (1) How often does bullying occur in internet chatrooms? (2) Who are the victims of bullying in internet chatrooms? (3) What are the determinants of victimization in internet chatrooms? A total of 1700 pupils from various German secondary schools participated in the study. Results revealed a strong relationship between victimization in school and victimization in internet chatrooms; school victims are significantly more often chat victims. Furthermore, the predictors of chat and school victimization show both commonalities (gender, self-concept, child-parent relationship) and differences (social integration, popularity, and bullying behavior).
Clinical application of NKT cell biology in type I (autoimmune) diabetes mellitus
Authors: Fletcher Marie T , Baxter Alan G
Published: February 2009

Type 1 natural killer T (NKT) cells are a population of CD1d‐restricted, regulatory T cells that exhibit various NK cell characteristics and rapidly produce cytokines on stimulation with glycolipid antigen. In type I diabetes (TID), NKT cells are thought to have a tolerogenic function, evidenced by NKT cell numerical and functional deficiencies in the nonobese diabetic (NOD) mouse, which when corrected, can ameliorate disease. The mechanisms by which NKT cells can mediate their immunosuppressive effects in NOD mice are still poorly understood, which makes successful clinical translation of NKT‐ cell‐based therapies challenging. However, new insights into the genetic control of NKT cell deficiencies have provided some understanding of the genes that may control NKT cell number and function, potentially offering a new avenue for assessing TID risk in humans. Here, we review the mechanisms by which NKT cells are thought to prevent TID, discuss the evidence for involvement of NKT cells in the regulation of human TID and examine the genetic control of NKT cell number and function. A greater understanding of these areas will increase the chances of successful clinical manipulation of NKT cells to prevent or treat TID.
Strategic Management Implications of a Consumer Value Perspective on Mobile Tv
Authors: Jarvenpaa Sirkka L , Loebbecke Claudia
Published: February 2009

Understanding of consumer experienced value in mobile services continues to be a strategic issue in the information systems literature. This is no different with Mobile TV that has become feasible with the convergence of telecommunication infrastructures, computer technology, and media content services. Mobile TV offerings are defined as real-time broadcast transmissions of content to mobile devices. We review mobile services literatures on consumer value creation and identify a gap in our understanding of how supplier firm's consumer-focused strategies may aid in consumption processes and thereby increase payments from consumers. We apply the perspective of consumer benefits experienced (CBE) to provide insight into how supplier firms may be able to increase consumer value. Value increases with greater consumer service-specific knowledge, and leveraging and complementing this knowledge across different offerings. Increased service-specific knowledge can also reduce demands on the consumer that can create important switching costs while increasing consumer-experienced value. This paper contributes to innovation research on mobile services.
The Impact of Geographic Context on E-Shopping Behavior
Authors: Ren Fang , Kwan Mei-Po
Published: February 2009

Recent studies have examined what factors affect the adoption of e-shopping (electronic shopping), why people adopt e-shopping, and what changes in activity–travel patterns will occur as a response to e-shopping. Very few studies to date, however, have investigated the impact of geographic context on people's e-shopping behavior. This study reexamines the explanatory factors that are related to people's e-shopping patterns through a study of the Columbus Metropolitan Area, OH. It focuses on the effect of accessibility to local shops and the residential context on the adoption of e-shopping and the frequency of buying online. Using an activity–Internet diary dataset, the results suggest that people with lower levels of accessibility to local shopping opportunities are more likely to engage in e-shopping, since the Internet enhances the efficiency of shopping by providing more product information and by eliminating the need of travel in the physical world. Further, people who live in areas with a white majority are more likely to adopt e-shopping. The magnitude of the impact of these context factors on e-shopping, however, is quite small.
Shadow Market Area for Air Pollutants
Authors: Meintrup David , Nam Chang Woon
Published: February 2009

This study identifies the shadow market area of air pollutants on the basis of the Gaussian plume model. Since pollutants are dispersed by wind, transport costs are irrelevant in its formation. Pollutant distribution on the ground level has an asymmetric bell shape in the wind direction. In addition to the linear functions, the exponential and quadratic shadow price functions are considered for the compensation of a health hazard to consumers, where the strict liability in the framework of the Coase theorem applies. The shadow market area for pollutants is shell shaped. This specific characteristic has an implication for market boundaries between polluters and their location decisions.
Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions
Authors: Björk Petra , Jin ShaoBo , Zhao Jian , Singh Om Prakash , Persson Jan-Olov , Hellman Ulf , Wieslander Lars
Published: February 2009

Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3′ end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.
A concentration-dependent endocytic trap and sink mechanism converts Bmper from an activator to an inhibitor of Bmp signaling
Authors: Kelley Rusty , Ren Rongqin , Pi Xinchun , Wu Yaxu , Moreno Isabel , Willis Monte , Moser Martin , Ross Malcolm , Podkowa Monika , Attisano Liliana , Patterson Cam
Published: February 2009

Bmper, which is orthologous to Drosophila melanogaster crossveinless 2, is a secreted factor that regulates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been postulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. In this paper, we demonstrate that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mechanism leads to the efficient degradation of Bmper and Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4-dependent Smad signaling. We also determined that Noggin and Gremlin, but not Chordin, trigger endocytosis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implications for Bmp signal regulation by secreted mediators.
Asymmetric enrichment of PIE-1 in the Caenorhabditis elegans zygote mediated by binary counterdiffusion
Authors: Daniels Brian R. , Perkins Edward M. , Dobrowsky Terrence M. , Sun Sean X. , Wirtz Denis
Published: February 2009

To generate cellular diversity in developing organisms while simultaneously maintaining the developmental potential of the germline, germ cells must be able to preferentially endow germline daughter cells with a cytoplasmic portion containing specialized cell fate determinants not inherited by somatic cells. In Caenorhabditis elegans, germline inheritance of the protein PIE-1 is accomplished by first asymmetrically localizing the protein to the germplasm before cleavage and subsequently degrading residual levels of the protein in the somatic cytoplasm after cleavage. Despite its critical involvement in cell fate determination, the enrichment of germline determinants remains poorly understood. Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve protein immobilization, intracellular compartmentalization, or localized protein degradation. Instead, our results support a heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in a stable protein gradient across the zygote at steady state.
One-dimensional topography underlies three-dimensional fibrillar cell migration
Authors: Doyle Andrew D. , Wang Francis W. , Matsumoto Kazue , Yamada Kenneth M.
Published: February 2009

Current concepts of cell migration were established in regular two-dimensional (2D) cell culture, but the roles of topography are poorly understood for cells migrating in an oriented 3D fibrillar extracellular matrix (ECM). We use a novel micropatterning technique termed microphotopatterning (μPP) to identify functions for 1D fibrillar patterns in 3D cell migration. In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome. We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.
The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria
Authors: Osman Christof , Haag Mathias , Potting Christoph , Rodenfels Jonathan , Dip Phat Vinh , Wieland Felix T. , Brügger Britta , Westermann Benedikt , Langer Thomas
Published: February 2009

Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.
MRN complex function in the repair of chromosomal Rag-mediated DNA double-strand breaks
Authors: Helmink Beth A. , Bredemeyer Andrea L. , Lee Baeck-Seung , Huang Ching-Yu , Sharma Girdhar G. , Walker Laura M. , Bednarski Jeffrey J. , Lee Wan-Ling , Pandita Tej K. , Bassing Craig H. , Sleckman Barry P.
Published: February 2009

The Mre11–Rad50–Nbs1 (MRN) complex functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) at postreplicative stages of the cell cycle. During HR, the MRN complex functions directly in the repair of DNA DSBs and in the initiation of DSB responses through activation of the ataxia telangiectasia-mutated (ATM) serine-threonine kinase. Whether MRN functions in DNA damage responses before DNA replication in G0/G1 phase cells has been less clear. In developing G1-phase lymphocytes, DNA DSBs are generated by the Rag endonuclease and repaired during the assembly of antigen receptor genes by the process of V(D)J recombination. Mice and humans deficient in MRN function exhibit lymphoid phenotypes that are suggestive of defects in V(D)J recombination. We show that during V(D)J recombination, MRN deficiency leads to the aberrant joining of Rag DSBs and to the accumulation of unrepaired coding ends, thus establishing a functional role for MRN in the repair of Rag-mediated DNA DSBs. Moreover, these defects in V(D)J recombination are remarkably similar to those observed in ATM-deficient lymphocytes, suggesting that ATM and MRN function in the same DNA DSB response pathways during lymphocyte antigen receptor gene assembly.
A novel myelin P0–specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy
Authors: Louvet Cédric , Kabre Beniwende G. , Davini Dan W. , Martinier Nicolas , Su Maureen A. , DeVoss Jason J. , Rosenthal Wendy L. , Anderson Mark S. , Bour-Jordan Hélène , Bluestone Jeffrey A.
Published: February 2009

Autoimmune-prone nonobese diabetic mice deficient for B7-2 spontaneously develop an autoimmune peripheral neuropathy mediated by inflammatory CD4+ T cells that is reminiscent of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. To determine the etiology of this disease, CD4+ T cell hybridomas were generated from inflamed tissue–derived CD4+ T cells. A majority of T cell hybridomas were specific for myelin protein 0 (P0), which was the principal target of autoantibody responses targeting nerve proteins. To determine whether P0-specific T cell responses were sufficient to mediate disease, we generated a novel myelin P0–specific T cell receptor transgenic (POT) mouse. POT T cells were not tolerized or deleted during thymic development and proliferated in response to P0 in vitro. Importantly, when bred onto a recombination activating gene knockout background, POT mice developed a fulminant form of peripheral neuropathy that affected all mice by weaning age and led to their premature death by 3–5 wk of age. This abrupt disease was associated with the production of interferon γ by P0-specific T cells and a lack of CD4+ Foxp3+ regulatory T cells. Collectively, our data suggest that myelin P0 is a major autoantigen in autoimmune peripheral neuropathy.
Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8
Authors: Handa Noriko , Terada Takaho , Kamewari Yuki , Hamana Hiroaki , Tame Jeremy R.H. , Park Sam‐Yong , Kinoshita Kengo , Ota Motonori , Nakamura Haruki , Kuramitsu Seiki , Shirouzu Mikako , Yokoyama Shigeyuki
Published: February 2009

The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG‐L in mammals, and GPI12 in yeast and protozoa, have N‐acetylglucosaminylphosphatidylinositol (GlcNAc‐PI) de‐N‐acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 Å resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted β‐sheet composed of six parallel β‐strands and one antiparallel β‐strand (with the strand order 3‐2‐1‐4‐5‐7‐6) sandwiched between six α‐helices. The N‐terminal five β‐strands and four α‐helices form an incomplete Rossmann fold‐like structure. The structure shares some similarity to the sugar‐processing enzymes with Rossmann fold‐like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)‐binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.
Catalytic and binding poly‐reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution
Authors: James Leo C. , Tawfik Dan S.
Published: February 2009

It is generally accepted that enzymes evolved via gene duplication of existing proteins. But duplicated genes can serve as a starting point for the evolution of a new function only if the protein they encode happens to exhibit some activity towards this new function. Although the importance of such catalytic promiscuity in enzyme evolution has been proposed, little is actually known regarding how common promiscuous catalytic activities are in proteins or their origins, magnitudes, and potential contribution to the survival of an organism. Here we describe a pattern of promiscuous activities in two completely unrelated proteins—serum albumins and a catalytic antibody (aldolase antibody 38C2). Despite considerable structural dissimilarities—in the shape of the cavities and the position of catalytic lysine residues—both active sites are able to catalyze the Kemp elimination, a model reaction for proton transfer from carbon. We also show that these different active sites can bind promiscuously an array of hydrophobic negatively charged ligands. We suggest that the basic active‐site features of an apolar pocket and a lysine residue can act as a primitive active site allowing these promiscuous activities to take place. We also describe, by modelling product formation at different substrate concentrations, how promiscuous activities of this kind— inefficient and rudimentary as they are—can provide a considerable selective advantage and a starting point for the evolution of new functions.
Free energies of protein decoys provide insight into determinants of protein stability
Authors: Vorobjev Yury N. , Hermans Jan
Published: February 2009

We have calculated the stability of decoy structures of several proteins (from the CASP3 models and the Park and Levitt decoy set) relative to the native structures. The calculations were performed with the force field–consistent ES/IS method, in which an implicit solvent (IS) model is used to calculate the average solvation free energy for snapshots from explicit simulations (ESs). The conformational free energy is obtained by adding the internal energy of the solute from the ESs and an entropic term estimated from the covariance positional fluctuation matrix. The set of atomic Born radii and the cavity‐surface free energy coefficient used in the implicit model has been optimized to be consistent with the all‐atom force field used in the ESs (cedar/gromos with simple point charge (SPC) water model). The decoys are found to have a consistently higher free energy than that of the native structure; the gap between the native structure and the best decoy varies between 10 and 15 kcal/mole, on the order of the free energy difference that typically separates the native state of a protein from the unfolded state. The correlation between the free energy and the extent to which the decoy structures differ from the native (as root mean square deviation) is very weak; hence, the free energy is not an accurate measure for ranking the structurally most native‐like structures from among a set of models. Analysis of the energy components shows that stability is attained as a result of three major driving forces: (1) minimum size of the protein‐water surface interface; (2) minimum total electrostatic energy, which includes solvent polarization; and (3) minimum protein packing energy. The detailed fit required to optimize the last term may underlie difficulties encountered in recovering the native fold from an approximate decoy or model structure.
The C‐terminal domain of biotin protein ligase from E. coli is required for catalytic activity
Authors: Chapman‐Smith Anne , Mulhern Terrence D. , Whelan Fiona , Cronan John E. , Wallace John C.
Published: February 2009

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl‐CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl‐5′‐AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site‐directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild‐type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C‐terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild‐type BirA. No function has been identified previously for the BirA C‐terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C‐terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25‐fold decrease in the affinity for ATP. Our data indicate that the C‐terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.
Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin‐like enzymes
Authors: Andreeva Natalia S. , Rumsh Lev D.
Published: February 2009

To elucidate the role of amino acid residues adjacent to the catalytic site of pepsin‐like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen‐bonded residues between the active site flap and the active carboxyls on ligand binding. These data suggest a new approach to understanding the role of residues around the catalytic site, which can assist the development of the catalytic reaction. The influence of groups adjacent to the active carboxyls is manifested by pepsin activity at pH 1.0. Some features of pepsin‐like enzymes and their mutants are discussed in the framework of the approach.
Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy
Authors: Chamberlain Aaron K. , Receveur Veronique , Spencer Andrew , Redfield Christina , Dobson Christopher M.
Published: February 2009

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non‐neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The 1H and 15N main‐chain amide chemical shifts of the I56T variant are very similar to those of the wild‐type protein, but those of the D67H variant are greatly altered for 28 residues in the β‐domain. This finding is consistent with the X‐ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild‐type protein in this region. The 1H–15N heteronuclear NOE values show that, with the exception of V121, every residue in the wild‐type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.
Competing protein:protein interactions are proposed to control the biological switch of the E coli biotin repressor
Authors: Weaver Larry H. , Kwon Keehwan , Beckett Dorothy , Matthews Brian W.
Published: February 2009

A model is suggested for the complex between the biotin repressor of Escherichia coli, BirA, and BCCP, the biotin carboxyl carrier protein to which BirA transfers biotin. The model is consistent with prior physical and biochemical studies. Measurement of transfer rates for variants of BirA with single‐site mutations in the proposed BirA:BCCP interface region also provides support. The unique feature of the proposed interaction between BirA and BCCP is that it uses the same β‐sheet region on the surface of BirA that the protein uses for homodimerization into a form competent to bind DNA. The resulting mutually exclusive protein:protein interfaces explain the novel feature of the BirA regulatory system, namely, that transcription of the genes involved in biotin synthesis is not determined by the level of biotin, per se, but by the level of unmodified BCCP. The model also provides a role for the C‐terminal domain of BirA that is structurally similar to an SH3 domain.
Identification of residues critical for metallo‐β‐lactamase function by codon randomization and selection
Authors: Materon Isabel C. , Palzkill Timothy
Published: February 2009

IMP‐1 β‐lactamase is a zinc metallo‐enzyme encoded by the transferable blaIMP‐1 gene, which confers resistance to virtually all β‐lactam antibiotics including carbapenems. To understand how IMP‐1 recognizes and hydrolyzes β‐lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the blaIMP‐1 gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP‐1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP‐1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild‐type enzyme.
Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3‐isopropyl 4‐(2‐chlorophenyl)‐1,4‐dihydro‐1‐ethyl‐2‐methyl‐pyridine‐3,5,6‐tricarboxylate
Authors: Oikonomakos Nikos G. , Tsitsanou Katerina E. , Zographos Spyros E. , Skamnaki Vicky T. , Goldmann Siegfried , Bischoff Hilmar
Published: February 2009

The effect of the potential antidiabetic drug (‐)(S)‐3‐isopropyl 4‐(2‐chlorophenyl)‐1,4‐dihydro‐1‐ethyl‐2‐methyl‐pyridine‐3,5,6‐tricarboxylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T‐state) to form the phosphorylated enzyme (GPa, more active, predominantly R‐state). Upon conversion of GPb to GPa, the N‐terminal tail (residues 5‐22), which carries the Ser14(P), changes its conformation into a distorted 310 helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T‐state GPb and produces further conformational changes, characteristic of a T′‐like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (‐AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 μM) with respect to glucose 1‐phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T‐state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 Å and 2.1 Å resolution, and refined to crystallographic R‐values of 0.179 (Rfree = 0.230) and 0.189 (Rfree = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N‐terminal tail occurs, while the loop of chain containing residues 192‐196 and residues 439‐499 shift to accommodate the ligand. Structural comparisons show that the T‐state GPa‐glucose‐W1807 structure is overall more similar to the T‐state GPb‐W1807 complex structure than to the GPa‐glucose complex structure, indicating that W1807 is able to transform GPa to the T′‐like state already observed with GPb. The structures provide a rational for the potency of the inhibitor and explain GPa allosteric inhibition of activity upon W1807 binding.
Probing the nature of interactions in SH2 binding interfaces–evidence from electrospray ionization mass spectrometry
Authors: Chung Evonne W. , Henriques Denise A. , Renzoni Debora , Morton Craig J. , Mulhern Terrence D. , Pitkeathly Maureen C. , Ladbury John E. , Robinson Carol V.
Published: February 2009

We have adopted nanoflow electrospray ionization mass spectrometry (ESI‐MS) and isothermal titration calorimetry (ITC) to probe the mechanism of peptide recognition by the SH2 domain from the Src family tyrosine kinase protein, Fyn. This domain is involved in the mediation of intracellular signal transduction pathways by interaction with proteins containing phosphorylated tyrosine (Y*) residues. The binding of tyrosyl phosphopeptides can mimic these interactions. Specificity in these interactions has been attributed to the interaction of the Y* and residues proximal and C‐terminal to it. Previous studies have established that for specific binding with Fyn, the recognition sequence consists of pTyr‐Glu‐Glu‐Ile. The specific interactions involve the binding of Y* with the ionic, and the Y* + 3 Ile residue with the hydrophobic binding pockets on the surface of the Fyn SH2 domain. In this work, a variation in the Y* + 3 residue of this high‐affinity sequence was observed to result in changes in the relative binding affinities as determined in solution (ITC) and in the gas phase (nanoflow ESI‐MS). X‐ray analysis shows that a feature of the Src family SH2 domains is the involvement of water molecules in the peptide binding site. Under the nanoflow ESI conditions, water molecules appear to be maintained in the Fyn SH2‐ligand complex. Compelling evidence for these molecules being incorporated in the SH2‐peptide interface is provided by the prevalence of the peaks assigned to water‐bound over the water‐free complex at high‐energy conditions. Thus, the stability of water protein‐ligand complex appears to be intimately linked to the presence of water.
Crystal structure of penicillin G acylase from the bro1 mutant strain of providencia rettgeri
Authors: Mcdonough Michael A. , Klei Herbert E. , Kelly Judith A.
Published: February 2009

Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild‐type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6‐bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6‐bromohexanamide. The X‐ray structure determination of this enzyme was undertaken to understand its altered specificity and to help in the design of site‐directed mutants with desired specificities. In this paper, the structure of the Bro1 penicillin G acylase has been solved at 2.5 Å resolution by molecular replacement. The R‐factor after refinement is 0.154 and R‐free is 0.165. Of the 758 residues in the Bro1 penicillin acylase heterodimer (α‐subunit, 205; β‐subunit, 553), all but the eight C‐terminal residues of the α‐subunit have been modeled based on a partial Bro1 sequence and the complete wild‐type P. rettgeri sequence. A tightly bound calcium ion coordinated by one residue from the α‐subunit and five residues from the β‐subunit has been identified. This enzyme belongs to the superfamily of Ntn hydrolases and uses Oγ of Serβ1 as the characteristic N‐terminal nucleophile. A mutation of the wild‐type Metα140 to Leu in the Bro1 acylase hydrophobic specificity pocket is evident from the electron density and is consistent with the observed specificity change for Bro1 acylase. The electron density for the N‐terminal Gln of the α‐subunit is best modeled by the cyclized pyroglutamate form. Examination of aligned penicillin acylase and cephalosporin acylase primary sequences, in conjunction with the P. rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase to accept charged β‐lactam R‐groups and to function as a cephalosporin acylase and thus be used in the manufacture of semi‐synthetic cephalosporins.
Three‐dimensional structures of enzyme‐substrate complexes of the hydroxynitrile lyase from hevea brasiliensis
Authors: Zuegg J. , Gruber K. , Gugganig M. , Wagner U. G. , Kratky C.
Published: February 2009

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb‐HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X‐ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80‐Oγ. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb‐HNL was redetermined at cryo‐temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb‐HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme‐catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.
Active site specificity of plasmepsin II
Authors: Westling Jennifer , Cipullo Patty , Hung Su‐Hwi , Saft Howard , Dame John B. , Dunn Ben M.
Published: February 2009

Members of the aspartic proteinase family of enzymes have very similar three‐dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys‐Pro‐Ile‐Leu‐Phe*Nph‐Ala/Glu‐Leu‐Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site‐directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.
Protein and ligand adaptation in a retinoic acid binding protein
Authors: Pattanayek Rekha , Newcomer Marcia E.
Published: February 2009

A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all‐trans and 9‐cis isomers of retinoic acid, as well as the synthetic retinoid (E)‐4‐[2‐(5,6,7,8)‐tetrahydro‐5,5,8,8‐tetramethyl‐2 napthalenyl‐1 propenyl]‐benzoic acid (TTNPB), a structural analog of all‐trans retinoic acid. The structure of ERABP with a mixture of all‐trans and 9‐cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all‐trans and 9‐cis isomers, the structures of all‐trans retinoic acid‐ERABP and 9‐cis retinoic acid ERABP were determined. Our results indicate that the all‐trans isomer of retinoic acid adopts an 8‐cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB‐ERABP is also reported herein. To accommodate this all‐trans analog, which cannot readily adopt a cis‐like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo‐proteins.