bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima
Authors: Welker Christine , Böhm Gerald , Schurig Hartmut , Jaenicke Rainer 
Published: October 2010

AbstractThermotoga maritima (Tm) expresses a 7 kDa monomeric protein whose 18 N‐terminal amino acids show 81% identity to N‐terminal sequences of cold shock proteins (Csps) from Bacillus caldolyticus and Bacillus stearothermophilus. There were only trace amounts of the protein in Thermotoga cells grown at 80°C. Therefore, to perform physicochemical experiments, the gene was cloned in Escherichia coli. A DNA probe was produced by PCR from genomic Tm DNA with degenerated primers developed from the known N‐terminus of TmCsp and the known C‐terminus of CspB from Bacillus subtilis. Southern blot analysis of genomic Tm DNA allowed to produce a partial gene library, which was used as a template for PCRs with gene‐and vector‐specific primers to identify the complete DNA sequence. As reported for other csp genes, the 5′ untranslated region of the mRNA was anomalously long; it contained the putative Shine–Dalgarno sequence. The coding part of the gene contained 198 bp, i.e., 66 amino acids. The sequence showed 61% identity to CspB from B. caldolyticus and high similarity to all other known Csps. Computer‐based homology modeling allowed the conclusion that TmCsp represents a β‐barrel similar to CspB from B. subtilis and CspA from E. coli.As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, over‐expression of the recombinant protein yielded authentic TmCsp with a molecular weight of 7,474 Da. This was in agreement with the results of analytical ultracentrifugation confirming the monomeric state of the protein. The temperature‐induced equilibrium transition at 87 °C exceeds the maximum growth temperature of Tm and represents the maximal Tm‐value reported for Csps so far.
pKa Calculations for class A β‐lactamases: Influence of substrate binding
Authors: Lamotte‐Brasseur Josette , Lounnas Valère , Raquet Xavier , Wade Rebecca C. 
Published: October 2010

Abstractβ‐Lactamases are responsible for bacterial resistance to β‐lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free β‐lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild‐type and three β‐lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri–Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a β‐lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the β‐lactam carboxylate on the pKa values of the active‐site lysines is also discussed.
Simplified methods for pKa and acid pH‐dependent stability estimation in proteins: Removing dielectric and counterion boundaries
Authors: Warwicker James 
Published: October 2010

AbstractMuch computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson–Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH‐dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye‐Hückel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad‐based discrepancy to measured pH‐dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH‐dependence of protein stability. The methods are applied to the relative pH‐dependent stabilities of the pore‐forming domains of colicins A and N. The results relate generally to the well‐known preponderance of surface ionizable groups with solvent‐mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large ΔpKas of a subset of ionizable groups with the overall robustness of the DH model.
A novel clan of zinc metallopeptidases with possible intramembrane cleavage properties
Authors: Lewis Alan P. , Thomas Pam J. 
Published: October 2010

AbstractComputer‐based database searching and protein multiple sequence alignment has identified a novel clan of zinc metallo‐peptidases, which, by phylogenetic analysis, has been shown to contain six subfamilies. The family is characterized by four common transmembrane segments and three conserved sequence motifs. The combination of topology analysis and motif identification has detected three potential Zn2+ coordinating residues. Only two of the sequences of this novel zinc metallopeptidase clan possess any functional annotation, one of which is able to cleave its substrate within a cytosol/transmembrane segment junction. A number of observations suggest that the remaining members of this novel clan may also cleave their substrates within transmembrane segments.
Bacterial rhodopsins: Evolution of a mechanistic model for the ion pumps
Authors: Stoeckenius Walther 
Published: October 2010

AbstractWalther Stoeckenius received a MD degree at the University of Hamburg, Germany in 1950. After 18 months of clinical work as an intern, he began postdoctoral work on the development of pox viruses at the Institute for Tropical Medicine in Hamburg using mainly electron microscopy techniques. After two years he moved as Assistant Professor to the Department of Pathology at the University of Hamburg and became Docent for Pathology in 1958. In addition to teaching and routine pathology work, he continued to use electron microscopy to explore the fine structure of cells and developed an interpretation of the triple‐layered appearance of membranes in electron micrographs in terms of molecular structure and the chemistry of osmium tetroxide fixation.In 1959 he obtained a position as Research Associate in Keith Porter's laboratory at Rockefeller University. This was changed after a few months to Assistant Professor and he stayed there, later as Associate Professor, for eight years. The work on membrane structure continued, and a model was developed that described the membrane as a lipid bilayer with embedded protein domains. In efforts to isolate such domains, the purple membrane and bacteriorhodopsin were discovered.In 1966, the lure of California became irresistible and Dr. Stoeckenius accepted a professorship at the University of California at San Francisco. The work on bacteriorhodopsin continued there with the emphasis changing from electron microscopy to spectroscopy and biochemical techniques. He is now Professor Emeritus there in the Department of Biochemistry and Biophysics and the Cardiovascular Research Institute.
Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains
Authors: Raffen Rosemarie , Dieckman Lynda J. , Szpunar Meredith , Wunschl Christine , Pokkuluri Phani R. , Dave Poras , Stevens Priscilla Wilkins , Cai Xiaoyin , Schiffer Marianne , Stevens Fred J. 
Published: October 2010

AbstractThe most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light‐chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant k4 light‐chain variable domains (VLs) derived from amyloidogenic light chains with a VL from a benign light chain. The amyloidogenic VLs were significantly less stable than the benign VL. Furthermore, only the amyloidogenic VLs formed fibrils under native conditions in an in vitro fibril formation assay. We used site‐directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic VLs on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign VL could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence‐specific features of the benign VL that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the VL β‐domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.
Mapping cyclic nucleotide‐induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases
Authors: Baichoo Noel , Heyduk Tomasz 
Published: October 2010

AbstractCyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830–10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe‐DTPA] 2), iron complexed to ethylenediaminediacetic acid ([Fe‐EDDA]), iron complexed to desferrioxamine mesylate ([Fe‐HDFO]+), and copper complexed to o‐phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydro‐phobicity. Binding of cAMPto CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe‐EDDA] and [Fe‐DTPA] 2− of CRP‐cAMP detected hypersensitivities in the DNA‐binding F α‐helix, the inter‐domain hinge, and the ends of the C α‐helix, which is involved in intersubunit interactions. [Fe‐EDDA] and [FeDTPA] 2− also detected reductions in cleavage in the D and E α‐helices, which are involved in DNA recognition. Cleavage by [Fe‐HDFO]+ of CRP‐cAMP detected hypersensitivities in β‐strand 8, the B α‐helix, as well as in parts of the F and C α‐helices. [Fe‐HDFO]+ also detected protections from cleavage in β‐strands 4 to 5 and their intervening loop, β‐strand 7, which is part of the nucleotide binding pocket, as well as in the D and E α‐helices. Cleavage by [(OP)2Cu]+ of CRP‐cAMP detected hypersensitivities in β‐strands 9 and 11 as well as in the D and E α‐helices. [(OP)2Cu]+ also detected protections in the C α‐helix, the interdomain hinge, and β‐strands 2–7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)2Cu]+, which detected minor protections in β‐strands 3–7, the interdomain hinge, and the C α‐helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction. Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone.
Structure in the channel forming domain of colicin E1 bound to membranes: The 402–424 sequence
Authors: SalwiSki Ukasz , Hubbell Wayne L. 
Published: October 2010

AbstractTo explore the structure of the pore‐forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402–424. Each mutant was reacted with a sulfhydryl‐specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406–416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402–420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408–R409, is located on the lipid‐exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.
Cloning, mutagenesis, and structural analysis of human pancreatic α‐amylase expressed in pichia pastoris
Authors: Rydberg Edwin H. , Sidhu Gary , Vo Hung C. , Hewitt Jeff , Ct Hlne C.F. , Wang Yili , Numao Shin , Macgillivray Ross T.A. , Overall Christopher M. , Brayer Gary D. , Withers Stephen G. 
Published: October 2010

AbstractHuman pancreatic &alpha;‐amylase &lpar;HPA&rpar; was expressed in the methylotrophic yeast Pichia pastoris and two mutants &lpar;D197A and D197N&rpar; of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry &lpar;ESI‐MS&rpar; to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI‐MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N‐linked oligosaccharide and yielded a protein product with a single N‐acetyl glucosamine &lpar;GlcNAc&rpar;, which could be crystallized. Solution of the crystal structure to a resolution of 2.0 &Aring; confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild‐type proteins were the same while the kcat&sol;Km values for D197A and D197N were 106&ndash;107 times lower than the wild‐type enzyme. The decreased kcat&sol;Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.
The mycobacterium tuberculosis reca intein can be used in an orftrap to select for open reading frames
Authors: Daugelat Sabine , Jacobs William R. 
Published: October 2010

AbstractThe DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self‐splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin‐resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self‐splicing properties of inteins (which depend on full‐length in‐frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non‐ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.
Protein structure comparison using iterated double dynamic programming
Authors: Taylor William R. 
Published: October 2010

AbstractA protein structure comparison method is described that allows the generation of large populations of high‐scoring alternate alignments. This was achieved by incorporating a random element into an iterative double dynamic programming algorithm. The maximum scores from repeated comparisons of a pair of structures converged on a value that was taken as the global maximum. This lay 15% over the score obtained from the single fixed (unrandomized) calculation. The effect of the gap penalty was observed through the shift of the alignment populations, characterized by their alignment length and root‐mean‐square deviation (RMSD). The best (lowest RMSD) values found in these populations provided a base‐line against which other methods were compared.
Grass: A server for the graphical representation and analysis of structures
Authors: Nayal Murad , Hitz Benjamin C. , Honig Barry 
Published: October 2010

AbstractGRASS (Graphical Representation and Analysis of Structures Server), a new web‐based server, is described. GRASS exploits many of the features of the GRASP program and is designed to provide interactive molecular graphics and quantitative analysis tools with a simple interface over the World‐Wide Web. Using GRASS, it is now possible to view many surface features of biological macromolecules on either standard workstations used in macromolecular analysis or personal computers. The result is a World‐Wide Web‐based, platform‐independent, easily used tool for macromolecular visualization and structure analysis.
CORA—Topological fingerprints for protein structural families
Authors: Orengo Christine Anne 
Published: October 2010

AbstractCORA is a suite of programs for multiply aligning and analyzing protein structural families to identify the consensus positions and capture their most conserved structural characteristics (e.g., residue accessibility, torsional angles, and global geometry as described by inter‐residue vectors/contacts). Knowledge of these structurally conserved positions, which are mostly in the core of the fold and of their properties, significantly improves the identification and classification of newly‐determined relatives. Information is encoded in a consensus three‐dimensional (3D) template and relatives found by a sensitive alignment method, which employs a new scoring scheme based on conserved residue contacts. By encapsulating these critical “core” features, templates perform more reliably in recognizing distant structural relatives than searches with representative structures.Parameters for 3D‐template generation and alignment were optimized for each structural class (mainly‐α, mainly‐β, α‐β), using representative superfold families. For all families selected, the templates gave significant improvements in sensitivity and selectivity in recognizing distant structural relatives. Furthermore, since templates contain less than 70% of fold positions and compare fewer positions when aligning structures, scans are at least an order of magnitude faster than scans using selected structures. CORA was subsequently tested on eight other broad structural families from the CATH database.Diagnostics plots are generated automatically and provide qualitative assistance for classifying newly determined relatives. They are demonstrated here by application to the large globin‐like fold family. CORA templates for both homologous superfamilies and fold families will be stored in CATH and used to improve the classification and analysis of newly determined structures.
Predicting the structures of 18 peptides using Geocore
Authors: Ishikawa Kohki , Yue Kaizhi , Dill Ken A. 
Published: October 2010

AbstractWe describe an extensive test of Geocore, an ab initio peptide folding algorithm. We studied 18 short molecules for which there are structures in the Protein Data Bank; chains are up to 31 monomers long. Except for the very shortest peptides, an extremely simple energy function is sufficient to discriminate the true native state from more than 108 lowest energy conformations that are searched explicitly for each peptide. A high incidence of native‐like structures is found within the best few hundred conformations generated by Geocore for each amino acid sequence. Predictions improve when the number of discrete ø/PS choices is increased.
Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure
Authors: Tsitsanou Katerina E. , Oikonomakos Nikos G. , Zographos Spyros E. , Skamnaki Vicky T. , Gregoriou Mary , Watson Kimberly A. , Johnson Louise N. , Fleet George W.J. 
Published: October 2010

AbstractThe effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X‐ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose‐1‐P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2‐methyl‐2,4‐pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and elethylene glycol as an uncompetitve inhibitor with respect to glucose‐1‐P. A parallel experimental investigation by X‐ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.