bluuebunny/crossref_metadata_2025 huggingface dataset:
A near‐native state on the slow refolding pathway of hen lysozyme
Authors: Kulkarni Sunita K. , Ashcroft Alison E. , Radford Sheena E. , Carey Michael , Masselos Dimitris , Robinson Carol V.
Published: October 2010

The refolding of four disulfide lysozyme (at pH 5.2, 20 °C) involves parallel pathways, which have been proposed to merge at a near‐native state. This species contains stable structure in the α‐and β‐domains but lacks a functional active site. Although previous experiments have demonstrated that the near‐native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 °C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths β‐domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near‐native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.
The signal transducer gp130: Solution structure of the carboxy‐terminal domain of the cytokine receptor homology region
Authors: Kernebeck Thomas , Pflanz Stefan , Heinrich Peter C. , Wollmer Axel , Grötzinger Joachim , Müller‐Newen Gerhard , Kurapkat Günther , Grzesiek Stephan , Scheek Ruud M. , Dijkstra Klaas
Published: October 2010

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin‐6‐type cytokines. It is a member of the cytokine‐receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine‐binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three‐dimensional structure of the carboxy‐terminal domain of this region was determined by multidimensional NMR. The domain consists of seven β‐strands constituting a fibronectin type III‐like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.
Protein‐protein recognition: An experimental and computational study of the R89K mutation in Raf and its effect on Ras binding
Authors: Zeng Jun , Simonson Thomas , Fridman Masha , Maruta Hiroshi , Treutlein Herbert R.
Published: October 2010

Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras–Raf binding affinity. We have investigated experimentally and computationally the Raf‐R89K mutation, which abolishes signaling in vivo. The binding to [γ‐35S]GTP‐Ras of a fusion protein between the Raf‐binding domain (RBD) of Raf and GST was reduced at least 175‐fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9 ± 1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein–protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 Å away, namely residues 37–41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2–3 specific water molecules. This analysis will provide insights into the large experimental database of Ras–Raf mutations.
Interaction of thioredoxins with target proteins: Role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2‐oxoacid dehydrogenase complexes
Authors: Bunik Victoria , Raddatz Günter , Bisswanger Hans , Lemaire Stephane , Meyer Yves , Jacquot Jean‐Pierre
Published: October 2010

The thioredoxin action upon the 2‐oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild‐type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin‐dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site‐directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2‐oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the α3/310 and a1 helices, the length of the α1 helix and the charges in the α2‐β3 and β4‐β5 linkers are found to correlate with the protein influence on the 2‐oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13–28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2‐oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.
Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non‐native secondary structure propensities
Authors: Cregut David , Serrano Luis
Published: October 2010

The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close–to–wild‐type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central α‐helix by the sequence of the second β‐hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one‐third of the secondary structure has been replaced by a non‐native one. Models of the mutants were built based on the three‐dimensional structure of the wild‐type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild‐type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.
Sequence specificity, statistical potentials, and three‐dimensional structure prediction with self‐correcting distance geometry calculations of β‐sheet formation in proteins
Authors: Zhu Hongyao , Braun Werner
Published: October 2010

A statistical analysis of a representative data set of 169 known protein structures was used to analyze the specificity of residue interactions between spatial neighboring strands in β‐sheets. Pairwise potentials were derived from the frequency of residue pairs in nearest contact, second nearest and third nearest contacts across neighboring β‐strands compared to the expected frequency of residue pairs in a random model. A pseudo‐energy function based on these statistical pairwise potentials recognized native β‐sheets among possible alternative pairings. The native pairing was found within the three lowest energies in 73% of the cases in the training data set and in 63% of β‐sheets in a test data set of 67 proteins, which were not part of the training set. The energy function was also used to detect tripeptides, which occur frequently in β‐sheets of native proteins. The majority of native partners of tripeptides were distributed in a low energy range. Self‐correcting distance geometry (SECODG) calculations using distance constraints sets derived from possible low energy pairing of β‐strands uniquely identified the native pairing of the β‐sheet in pancreatic trypsin inhibitor (BPTI). These results will be useful for predicting the structure of proteins from their amino acid sequence as well as for the design of proteins containing β‐sheets.
Estimation of the number of α‐helical and β‐strand segments in proteins using circular dichroism spectroscopy
Authors: Sreerama Narasimha , Venyaminov Sergei Y.U. , Woody Robert W.
Published: October 2010

A simple approach to estimate the number of α‐helical and β‐strand segments from protein circular dichroism spectra is described. The α‐helix and β‐sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given α‐helix or β‐strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per α‐helix and two residues per β‐strand may be considered distorted in proteins. The number of α‐helical and β‐strand segments and their average length in a given protein were estimated from the fraction of distorted α‐helix and β‐strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.
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

Thermotoga 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

β‐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

Much 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

Computer‐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

Walther 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

The 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

CyclicAMP 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

To 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

Human pancreatic α‐amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI‐MS) 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 (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 Å 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/Km values for D197A and D197N were 106–107 times lower than the wild‐type enzyme. The decreased kcat/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

The 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

A 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

GRASS (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

CORA 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

We 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

The 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.
Helicity of α(404–451) and β(394–445) tubulin C‐terminal recombinant peptides
Authors: Jimenez M. Angeles , Evangelio Juan A. , Aranda Carlos , Lopez‐Brauet Adamari , Andreu David , Rico Manuel , Lagos Rosalba , Andreu Jose M. , Monasterio Octavio
Published: October 2010

We have investigated the solution conformation of the functionally relevant C‐terminal extremes of α‐ and β‐tubulin, employing the model recombinant peptides RL52a3 and RL33b6, which correspond to the amino acid sequences 404‐451(end) and 394–445(end) of the main vertebrate isotypes of α‐ and β‐tubulin, respectively, and synthetic peptides with the α‐tubulin(430–443) and β‐tubulin(412–431) internal sequences. α(404–451) and β(394–445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of β(394–445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409‐Glu410 and Tyr422‐Gln423‐Gln424, defining the proteolysis resistant segment 410–422, which corresponds to the central part of the predicted β‐tubulin C‐terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)‐induced markedly helical CD spectra in α(404–451) and β(394–445). A substantial part of the helicity of β(394–445) was found to be in the CD spectrum of the shorter peptide β(412–431) with TFE. Two‐dimensional 1H‐NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational CαH shifts) in 30% TFE permitted to conclude that about 25% of α(404–451) and 40% of β(394–451) form well‐defined helices encompassing residues 418–432 and 408–431, respectively, flanked by disordered N‐ and C‐segments. The side chains of β(394–451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both α and β chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of α(404–451) is the same as that described in the electron crystallographic model structure of αβ‐tubulin, while in β(394–451) it extends for nine residues more, supporting the possibility of a functional coil → helix transition at the C‐terminus of β‐tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.
Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: A calorimetric and spectroscopic study
Authors: Poklar Nataša , Petrovčič Nina , Oblak Miha , Vesnaver Gorazd
Published: October 2010

The effect of methylurea, N,N'‐dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near‐ and far‐UV range, it has been observed that the tertiary structure of RNase A melts at about 3 °C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two‐state approximation. The far‐UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 °C. The concentration dependence of the preferential solvation change Δγ23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.
De novo design of a monomeric three‐stranded antiparallel β‐sheet
Authors: Alba Eva De , Santoro Jorge , Rico Manuel , Jiménez M. Angeles
Published: October 2010

Here we describe the NMR conformational study of a 20‐residue linear peptide designed to fold into a monomeric three‐stranded antiparallel β‐sheet in aqueous solution. Experimental and statistical data on amino acid β‐turn and β‐sheet propensities, cross‐strand side‐chain interactions, solubility criteria, and our previous experience with β‐hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C‐NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12‐residue peptides encompassing the N‐ and C‐segments of the designed sequence indicates that the 20‐residue peptide folds into the expected conformation. Assuming a two‐state model, the exchange kinetics between the β‐sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil‐β‐sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in β‐sheet formation and stability.
Selection of antibody probes to correlate protein sequence domains with their structural distribution
Authors: Valle Mikel , Muñoz Manuel , Kremer Leonor , Valpuesta Jose M. , Martínez‐A Carlos , Carrascosa Jose L. , Albar Juan P.
Published: October 2010

We propose a new approach that permits correlation of specific domains defined by their primary sequence with their location in the structure of complex macromolecular aggregates. It is based on the combination of well‐established structural analysis methods that incorporate the use of overlapping peptides on cellulose membranes for the isolation and purification of specific antibodies from a polyclonal antiserum. Monospecific antibodies to the connector protein of bacteriophage ø29 were isolated from polyclonal antisera using a new development of the spotscan method. These antibodies can be purified in quantities that allow antigenicity testing in enzyme‐linked immunosorbent assays, Western blotting and immunoprecipitations, demonstrating the specificity of this isolation procedure. This approach has allowed us to generate direct antibody probes for immunoelectron microscopy mapping of different connector protein domains in a low resolution three‐dimensional epitope map.
Folding of beta‐sandwich proteins: Three‐state transition of a fibronectin type III module
Authors: Cota Ernesto , Clarke Jane
Published: October 2010

An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three‐state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two‐state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.
A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data
Authors: Sillen Alain , Díaz José Fernando , Engelborghs Yves
Published: October 2010

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca1+ or Mg1+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha‐ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.
Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry
Authors: Louie Donna R , Gloor Kristen K. , Galasinski Scott C. , Resing Katheryn A. , Ahn Natalie G.
Published: October 2010

High mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG‐14 and ‐17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full‐length masses demonstrated mono‐, di‐, and triphosphorylation of HMG‐14 and mono‐ and diphosphorylation of HMG‐17 from OA treated cells, whereas HMG‐14 and ‐17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG‐17, and Ser20 and Ser24 in HMG‐14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG‐14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG‐14 and ‐17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG‐14 and ‐7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei.
Crystal structures of two mutants (K206Q, H207E) of the N‐lobe of human transferrin with increased affinity for iron
Authors: Yang Amy H.‐W. , Macgillivray Ross T.A. , Chen Jie , Luo Yaoguang , Wang Yili , Brayer Gary D. , Mason Anne B. , Woodworth Robert C. , Murphy Michael E.P.
Published: October 2010

The X‐ray crystallographic structures of two mutants (K206Q and H207E) of the N‐lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 Å, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H‐bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.