bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissection of the gene of the bifunctional PGK‐TIM fusion protein from the hyperthermophilic bacterium Thermotoga maritima: Design and characterization of the separate triosephosphate isomerase
Authors: Beaucamp Nicola , Hofmann Alexandra , Kellerer Barbara , Jaenicke Rainer 
Published: February 2009

AbstractTriosephosphate isomerase (TIM), from the hyperthemophilic bacterium Thermotoga maritima, has been shown to be covalently linked to phosphoglycerate kinase (PGK) forming a bifunctional fusion protein with TIM as the C‐terminal portion of the subunits of the tetrameric protein (Schurig et al., EMBO J 14:442‐451, 1995). To study the effect of the anomalous state of association on the structure, stability, and function of Thermotoga TIM, the isolated enzyme was cloned and expressed in Escherichia coli, and compared with its wild‐type structure in the PGK‐TIM fusion protein. After introducing a start codon at the beginning of the tpi open reading frame, the gene was expressed in E.c.BL21(DE3)/ pNBTIM. The nucleotide sequence was confirmed and the protein purified as a functional dimer of 56.5 kDa molecular mass. Spectral analysis, using absorption, fluorescence emission, near‐ and far‐UV circular dichroism spectroscopy were used to compare the separated Thermotoga enzyme with its homologs from mesophiles. The catalytic properties of the enzyme at ∼ 80°C are similar to those of its mesophilic counterparts at their respective physiological temperatures, in accordance with the idea that under in vivo conditions enzymes occupy corresponding states. As taken from chaotropic and thermal denaturation transitions, the separated enzyme exhibits high intrinsic stability, with a half‐concentration of guanidinium‐chloride at 3.8 M, and a denaturation half‐time at 80° C of 2 h. Comparing the properties of the TIM portion of the PGK‐TIM fusion protein with those of the isolated recombinant TIM, it is found that the fusion of the two enzymes not only enhances the intrinsic stability of TIM but also its catalytic efficiency.
Evidence for two conformational states of thioredoxin reductase from Escherichia coli: Use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation
Authors: Mulrooney Scott B. , Williams Charles H. 
Published: February 2009

AbstractThioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two‐stranded β sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox‐active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66° relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800‐816), placing either the redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active‐site mutant. The flavin fluorescence of this mutant is only 7% that of wild‐type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active‐site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5‐fold increase in fluorescence. Titration of the PMA‐treated mutant with the nonreducing NADP(H) analogue, 3‐aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild‐type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA‐treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.
Conformationally driven protease‐catalyzed splicing of peptide segments: V8 protease‐mediated synthesis of fragments derived from thermolysin and ribonuclease A
Authors: Kumaran Sangaralingam , Datta Debjani , Roy Rajendra P. 
Published: February 2009

AbstractWe have studied the conformation as well as V8 protease‐mediated synthesis of peptide fragments, namely amino acid residues 295‐316 (TC‐peptide) of thermolysin and residues 1‐20 (S‐peptide) of ribonuclease A, to examine whether “conformational trapping” of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent‐mediated cooperative helix formation in TC‐peptide with attainment of about 30‐35% helicity in the presence of 40% 1‐propanol and 2‐propanol solutions at pH 6 and 4°C. The thermal melting profiles of TC‐peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC‐peptide from a 1:1 mixture of the noninteracting complementary fragments (TC295‐302 and TC303‐316) in the presence of the above cosolvents at pH 6 and 4°C. In contrast, V8 protease did not catalyze the ligation of S1‐9 and S10‐20, although S‐peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S‐peptide (SA‐peptide) in which residues 10‐14 were substituted (RQHMD → VAAAK). While S‐peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA‐peptide displayed predominantly β‐sheet conformation. SA‐peptide showed enhanced resistance to proteolysis as compared with S‐peptide. Thus, failure of semisynthesis of S‐peptide may be a consequence of high flexibility around the 9‐10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease‐mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.
Three‐dimensional solution structure of β cryptogein, a β elicitin secreted by a phytopathogenic fungus phytophthora cryptogea
Authors: Fefeu Sandrine , Bouaziz Serge , Guittet Eric , Huet Jean‐Claude , Pernollet Jean‐Claude 
Published: February 2009

AbstractCryptogein belongs to a new family of 10‐kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive‐like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of β cryptogein from Phytophthoru cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE‐derived distance restraints and 40 dihedral angle restraints obtained from 3JHNHα couplings. The RMS deviation from the mean structure is 0.87 ± 0.14 Å for backbone atoms and 1.34 ± 0.14 Å for all the non‐hydrogen atoms of residues 2 to 98. The structure of β cryptogein reveals a novel protein fold, with five helices and a double‐stranded β‐sheet facing an Ω‐loop. One edge of the β‐sheet and the adjacent face of the Ω‐loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X‐ray structure, with slight differences lightly due to the crystal packing.
Anatomy of a conformational transition of β‐strand 6 in soybean P‐amylase caused by substrate (or inhibitor) binding to the catalytical site
Authors: Pujadas Gerard , Palau Jaume 
Published: February 2009

AbstractA computational study of the five soybean β‐amylase X‐ray structures reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the β‐strand 6 (residues 341‐343) in the (β/α)8 molecular scaffold. Backbone distortions that involve considerable changes in the ø and ψ angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA‐layer (at the C‐terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand‐binding process, there is a negative enthalpy change for these residues (—27 and —34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed “stability‐function” hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452‐456). They are also in good agreement with other experimental results in the literature that describe the role of the 341‐343 segment in β‐amylase activity. Site‐directed mutagenesis focused on these residues could be useful for undertaking functional studies of β‐amylase.
Pilin C‐terminal peptide binds asialo‐GM1 in liposomes: A 2H‐NMR study
Authors: Jones David H. , Barber Kathryn R. , Grant Chris W. M. , Hodges Robert S. 
Published: February 2009

AbstractWideline 2H‐NMR observations are described demonstrating the interaction of a synthetic peptide (PAK), representing residues 128‐144 of the binding domain of pilin surface protein from Pseudomonas aeruginosa, with a complex glycosphingolipid thought to be its natural receptor. The receptor glycolipid (asialo‐GM1) carried 2H probe nuclei on the terminal and next‐to‐terminal carbohydrate residues and was present as a minor component in fluid phosphatidylcholine liposomes. The peptide induced spectral changes that could be understood as arising from receptor motional changes, without receptor immobilization on the NMR time scale of 104 s−1. Spectral effects were reversed by reduction of the single peptide disulfide bond—a structural feature previously shown to be a determinant of PAK conformation (Campbell AP, McInnes C, Hodges RS, Sykes BD. 1995. Biochemistry 34:6255‐16268). This is the first demonstration of PAK interaction with its epithelial cell receptor in liposomes.
Preliminary structural studies on the multi‐ligand‐binding domain of the transcription activator, BmrR, from bacillus subtilis
Authors: Zheleznova Ekaterina E. , Brennan Richard G. , Markham Penelope N. , Neyfakh Alexander A. 
Published: February 2009

AbstractIn the bacterium Bacillus subtilis, the DNA‐binding regulatory protein, BmrR, activates transcription from the multidrug transporter gene, bmr, after binding either rhodamine or tetraphenylphosphonium. These two compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR family of transcription activators but differs from the other family members in its ability to bind unrelated small molecule activators. As an initial step in the elucidation of the mechanism by which BmrR recognizes rhodamine and tetraphenylphosphonium and activates transcription, we have crystallized the 144‐amino acid‐residue carboxy terminal dimerization/ ligand‐binding domain of the BmrR, named the BRC (BmrR C‐terminus). Tetragonal crystals of ligand‐free BRC take the space group P41212, or its enantiomorph P43212, with unit cell dimensions a = b = 76.3 Å, c = 96.0 Å, α = β = γ = 90°. Diffraction is observed to at least 2.7 Å resolution at room temperature. In addition, we determined the secondary structure content of ligand‐free and rhodamine‐bound BRC by circular dichroism. In the ligand‐free form, BRC has considerable β‐sheet content (41%) and little α‐helix structure (13%). After BRC binds rhodamine, its β‐sheet content increases to 47% while the α‐helix structure decreases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the elucidation of the recognition and efflux mechanisms of Bmr and other bacterial multidrug transporters.
Knowledge‐based model of a glucosyltransferase from the oral bacterial group of mutans streptococci
Authors: Devulapalle Kumari S. , Goodman Steven D. , Gao Qian , Hemsley Ann , Mooser Gregory 
Published: February 2009

AbstractMutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of α‐amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge‐based interactive algorithm, MACAW, which provided significant level of homology with α‐amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/α‐amylase residues conserved in all but one α‐amylase invariant residues. Site‐directed mutagenesis of the three GTF catalytic residues are homologous with the α‐amylase catalytic triad. The glucosyltransferases are members of the 4/7‐superfamily that have a (β/α)8‐barrel structure and belong to family 13 of the glycohydralases.
Structural characterization of human hemoglobin crosslinked by bis(3,5‐dibromosalicyl) fumarate using mass spectrometric techniques
Authors: Yu Zhonghua , Friso Giulia , Miranda Jj , Burlingame Alma L. , Patel Mehul J. , Lo‐Tseng Thomas , Moore Edwin G. 
Published: February 2009

AbstractDiaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric‐based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5‐dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size‐exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP‐HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin β‐chain, and two hemoglobin α‐chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.
Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan‐synthase β chain
Authors: Gast Klaus , Mueller‐Frohne Marlies , Chaffotte Alain F. , Guillou Yvonne , Hodges Maria , Cadieux CÉLine , Goldberg Michel E. , Zirwer Dietrich , Damaschun Gregor 
Published: February 2009

AbstractThe isolated, 101‐residue long C‐terminal (so called F2) fragment of the β chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far‐UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60°C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.
α‐Hemolysin, γ‐hemolysin, and leukocidin from Staphylococcus aureus: Distant in sequence but similar in structure
Authors: Gouaux Eric , Hobaugh Michael , Song Langzhou 
Published: February 2009

Abstractα‐Hemolysin from Staphylococcus aureus assembles from a water‐soluble, monomeric species to a membrane‐bound heptamer on the surface of target cells, creating water‐filled channels that lead to cell death and lysis. Staphylococcus aureus also produces the γ‐hemolysin and leukocidin toxins, which function as two component toxins in the disruption and lysis of erythrocytes and leukocytes. Analysis of the aligned sequences of α‐hemolysin, γ‐hemolysin, and leukocidin in the context of the α‐hemolysin heptamer structure supports the conclusion that even though the level of sequence identity between α‐hemolysin and the γ‐hemolysin and leukocidin toxins is in the so‐called twilight zone, the three‐dimensional structures of the protomers are probably conserved. By analogy with α‐hemolysin, γ‐hemolysin and leukocidin may also form oligomeric, transmembrane channels in which an anti‐parallel β‐barrel constitutes the primary membrane‐embedded domain.
Crystal structures of the cadmium‐ and mercury‐substituted metallo‐β‐lactamase from Bacteroides fragilis
Authors: Concha Nestor O. , Herzberg Osnat , Rasmussen Beth A. , Bush Karen 
Published: February 2009

AbstractThe metallo‐β‐lactamases require zinc or cadmium for hydrolyzing β‐lactam antibiotics and are inhibited by mercurial compounds. To date, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn2+‐bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the β‐lactam ring. To study the metal coordination further, the crystal structures of a Cd2+‐bound enzyme and of an Hg2+‐soaked zinc‐containing enzyme have been determined at 2.I Å and 2.7 Å, respectively. Given the diffraction resolution, the Cd2+‐bound enzyme exhibits the same active‐site architecture as that of the Zn2+‐bound enzyme, consistent with the fact that both forms are enzymatically active. The 10‐fold reduction in activity of the Cd2+‐bound molecule compared with the Zn2+‐bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg2+‐bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2‐Zn2+‐bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 Å away from Zn1 and 3.9 Å away from the site where Zn2 is located in the 2‐Zn2+‐bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.
Hinge bending within the cytokine receptor superfamily revealed by the 2.4 Å crystal structure of the extracellular domain of rabbit tissue factor
Authors: Muller Yves A. , Kelley Robert F. , De Vos Abraham M. 
Published: February 2009

AbstractTissue factor (TF), a member of the cytokine receptor superfamily, is the obligate cofactor of coagulation factor VIIa (FVIIa), and has a pivotal role in initiating the extrinsic pathway of blood coagulation through formation of the TF‐FVIIa complex. The crystal structure of the extracellular portion of rabbit TF has been solved at 2.35 Å resolution and refined to a crystallographic R‐value of 19.1% (free R‐value, 27.7%). Like the human homologue, the extracellular portion consists of two fibronectin type III domains connected by a short α‐helical segment. Unexpectedly, the two molecules in the crystallographic asymmetric unit differ in their relative domain‐domain orientation, revealing unsuspected hinge motion consisting of a rotation of about 12.7° around an axis intersecting the linker segment at residue 106. Superposition of rabbit tissue factor with free and bound human tissue factor allows for the detection of an identical, albeit smaller, hinge motion in human TF induced upon binding of FVIIa. This raises the possibility that a very similar hinge axis may be present in other members of the cytokine receptor superfamily.
A thioredoxin fusion protein of VanH, a D‐lactate dehydrogenase from Enterococcus faecium: Cloning, expression, purification, kinetic analysis, and crystallization
Authors: Stoll Vincent S. , Manohar A. Vaito , Gillon Wanda , Macfarlane Emma L. A. , Hynes Rosemary C. , Pai Emil F. 
Published: February 2009

AbstractThe gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D‐lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on kcat/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of ∼ 3.5 μ, 19.0 μ, and 1.5 μ, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408‐10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP‐ribose, NAD +, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash‐frozen crystals of the tetragonal form diffracted to 3.0 Å resolution at a synchrotron radiation source.
Production, crystallization, and preliminary X‐ray analysis of the human MHC class Ib molecule HLA‐E
Authors: O'callaghan Christopher A. , McMichael Andrew J. , Bell John I. , Blundell Charlie D. , Jakobsen Bent K. , Willcox Benjamin E. , Tormo José , Stuart David I. , Jones E. Yvonne 
Published: February 2009

AbstractHLA‐E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA‐E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA‐E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA‐E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA‐E forms a stable complex with β2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 Å with space group P3I21 and unit cell dimensions a = 182.2 Å, b = 182.2 Å, c = 88.4 Å.