bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stability of yeast iso‐1‐ferricytochrome c as a function of pH and temperature
Authors: Cohen David S. , Pielak Gary J. 
Published: February 2009

AbstractAbsorbance‐detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso‐1‐ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2‐state model. Values for free energy (ΔGD), enthalpy (ΔHD), and entropy (ΔSD) of denaturation were determined as functions of pH and temperature. The value of ΔGD at 300 K, pH 4.6, is 5.1 ± 0.3 kcal mol−1. The change in molar heat capacity upon denaturation (ΔCp), determined by the temperature dependence of ΔHD as a function of pH (1.37 ± 0.06 kcal mol−1 K−1), agrees with the value determined by differential scanning calorimetry. pH‐dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a ΔHD of 1.30 kcal mol−1 residue−1 at 373.6 K and a ΔSD of 4.24 cal mol−1 K−1 residue−1 at 385.2 K. These values agree with those for other single‐domain, globular proteins.
Determination of the structure of the DNA binding domain of γΔ resolvase in solution
Authors: Liu Tun , Derose Eugene F. , Mullen Gregory P. 
Published: February 2009

AbstractThe DNA binding domain (DBD) of γΔ resolvase (residues 141–183) is responsible for the interaction of this site‐specific DNA recombinase with consensus site DNA within the γΔ transposable element in Escherichia coli. Based on chemical‐shift comparisons, the proteolytically isolated DBD displays side‐chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309–16315). The structure of the DBD in solution has been determined using restraints obtained from 2‐dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 ϕ and X1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data. With respect to the computed average structure, these conformers display an RMS deviation of 0.67 Å for the heavy backbone atoms and 1.49 Å for all heavy atoms within residues 149–180. The DBD consists of 3 α‐helices comprising residues D149–Q157, S162–T167, and R172–N183. Helix‐2 and helix‐3 form a backbone fold, which is similar to the canonical helix‐turn‐helix motif. The conformation of the NH2‐terminal residues, G141–R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix‐1 and helix‐3 cross with a right‐handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix‐3 in the major groove and the amino‐terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by γΔ resolvase and provides initial structure‐function comparisons among the divergent DBDs of related resolvases and invertases.
Deuterium exchange of α‐helices and β‐sheets as monitored by electrospray ionization mass spectrometry
Authors: Wagner David S. , Melton Laura G. , Yan Yibing , Erickson Bruce W. , Anderegg Robert J. 
Published: February 2009

AbstractDeuterium exchange was monitored by electrospray ionization mass spectrometry (ESI‐MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several α‐helical peptides and β‐sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, α‐helical, or β‐sheet structure. For 3 isomeric 31‐residue α‐helical peptides, the number of slowly exchanging hydrogens as measured by ESI‐MS in 50% CF3CD2OD (pD 9.5) provided estimates of their α‐helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of β‐sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD3OD/0.5% CD3CO2D (pD 3.8) as measured by ESI‐MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI‐MS requires much less sample (1–50 μg), much shorter analysis time (10–90 min), and no chemical quenching of the exchange reaction.
Recognition of related proteins by iterative template refinement (ITR)
Authors: Yi Tau‐Mu , Lander Eric S. 
Published: February 2009

AbstractPredicting the structural fold of a protein is an important and challenging problem. Available computer programs for determining whether a protein sequence is compatible with a known 3‐dimensional structure fall into 2 categories: (1) structure‐based methods, in which structural features such as local conformation and solvent accessibility are encoded in a template, and (2) sequence‐based methods, in which aligned sequences of a set of related proteins are encoded in a template. In both cases, the programs use a static template based on a predetermined set of proteins. Here, we describe a computer‐based method, called iterative template refinement (ITR), that uses templates combining structure‐based and sequence‐based information and employs an iterative search procedure to detect related proteins and sequentially add them to the templates. Starting from a single protein of known structure, ITR performs sequential cycles of database search to construct an expanding tree of templates with the aim of identifying subtle relationships among proteins. Evaluating the performance of ITR on 6 proteins, we found that the method automatically identified a variety of subtle structural similarities to other proteins. For example, the method identified structural similarity between arabinose‐binding protein and phosphofructokinase, a relationship that has not been widely recognized.
A new family of carbon‐nitrogen hydrolases
Authors: Bork Peer , Koonin Eugene V. 
Published: February 2009

AbstractUsing computer methods for database search and multiple alignment, statistically significant sequence similarities were identified between several nitrilases with distinct substrate specificity, cyanide hydratases, aliphatic amidases, β‐alanine synthase, and a few other proteins with unknown molecular function. All these proteins appear to be involved in the reduction of organic nitrogen compounds and ammonia production. Sequence conservation over the entire length, as well as the similarity in the reactions catalyzed by the known enzymes in this family, points to a common catalytic mechanism. The new family of enzymes is characterized by several conserved motifs, one of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis.
Protein identification in DNA databases by peptide mass fingerprinting
Authors: James Peter , Quadroni Manfredo , Carafoli Ernesto , Gonnet Gaston 
Published: February 2009

AbstractProteins can be identified using a set of peptide fragment weights produced by a specific digestion to search a protein database in which sequences have been replaced by fragment weights calculated for various cleavage methods. We present a method using multidimensional searches that greatly increases the confidence level for identification, allowing DNA sequence databases to be examined. This method provides a link between 2‐dimensional gel electrophoresis protein databases and genome sequencing projects. Moreover, the increased confidence level allows unknown proteins to be matched to expressed sequence tags, potentially eliminating the need to obtain sequence information for cloning. Database searching from a mass profile is offered as a free service by an automatic server at the ETH, Zürich. For information, send an electronic message to the address cbrg@inf.ethz.ch with the line: help mass search, or help all.
Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone
Authors: Shastry M. C. R. , Agashe Vishwas R. , Udgaonkar Jayant B. 
Published: February 2009

AbstractThe fluorescence‐monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 °C, have been quantitatively analyzed using a 3‐state mechanism: US → UF →N. US and UF are slow‐refolding and fast‐refolding unfolded forms of barstar, and N is the native protein. US and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47‐Pro 48 bond. The 3‐state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3‐state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio US:UF (K21) and the values of the rate constants of the US → UF and UF → US reactions, k12 and k21 respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 ± 0.1, and k12 and k21 have values of 5.3 × 10−3 s−1 and 11.2 × 10−3 s−1, respectively. Double‐jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3‐state analysis and thereby the validity of the 3‐state model. The 3‐state model does not account for the kinetics of folding in the pretransition region, where folding occurs by 2 parallel pathways, UF → N, and US → IN → N, and IN is a native‐like intermediate. The rate constants of the UF → N and US → IN reactions are both similar, with values of 37 s−1 in water. The IN → N reaction, which involves the same trans‐cis isomerization process as the US → UF reaction, occurs with a rate constant of 16 × 10−3 s−1 and is independent of GdnHCl concentration. Thus, trans‐cis isomerization occurs 3 times faster in the folding intermediate than in the unfolded state.
Refined structure of dimeric diphtheria toxin at 2.0 Å resolution
Authors: Bennett M. J. , Choe S. , Eisenberg David 
Published: February 2009

AbstractThe refined structure of dimeric diphtheria toxin (DT) at 2.0 Å resolution, based on 37,727 unique reflections (F > 1σ(F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3′‐5′ uridine 3′ monophosphate (ApUp), and 405 well‐ordered water molecules. The 2.0‐Å refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor‐binding domains and is different from the Rossmann dinucleotide‐binding fold. ApUp is bound in part by a long loop (residues 34–52) that crosses the active site. Several residues in the active site were previously identified as NAD‐binding residues. Glu 148, previously identified as playing a catalytic role in ADP‐ribosylation of elongation factor 2 by DT, is about 5 Å from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an “open” structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by “domain swapping” to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.
Refined structure of monomelic diphtheria toxin at 2.3 Å resolution
Authors: Bennett M. J. , Eisenberg David 
Published: February 2009

AbstractThe structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 Å resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3′‐5′ uridine 3′ monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the “open” monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000–0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main‐chain conformations of 8 residues acting as a hinge to “open” or “close” the receptor‐binding (R) domain, and (2) a possible receptor‐docking site, a β‐hairpin loop protruding from the R domain containing residues that bind the cell‐surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5‐step structure‐based mechanism of intoxication: (1) proteolysis of a disulfide‐linked surface loop (residues 186–201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a β‐hairpin loop protruding from the R domain to the DT receptor, leading to receptor‐mediated endocytosis; (3) low pH‐triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP‐ribosylation of elongation factor 2.
Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor
Authors: Orlandini Maurizio , Santucci Annalisa , Tramontano Anna , Neri Paolo , Oliviero Salvatore 
Published: February 2009

AbstractIn this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3‐dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody‐Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site‐directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.
Epitope mapping of the gastrin‐releasing peptide/anti‐bombesin monoclonal antibody complex by proteolysis followed by matrix‐assisted laser desorption ionization mass spectrometry
Authors: Papac Damon I. , Hoyes John , Tomer Kenneth B. 
Published: February 2009

AbstractWe have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen‐antibody complex followed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI/TOF). A mouse anti‐bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin‐releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen‐antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen‐antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed‐phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti‐bombesin monoclonal antibody was shown to comprise the last 7–8 residues (HWAVGHLM‐NH2) of GRP.
Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa
Authors: Norin Martin , Haeffner Fredrik , Achour Adnane , Norin Torbjörn , Hult Karl 
Published: February 2009

AbstractThe substrate‐binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme.Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme‐substrate transition‐state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.
Anatomy of an engineered NAD‐binding site
Authors: Mittl P.R.E. , Berry A. , Scrutton N. S. , Perham R. N. , Schulz G. E. 
Published: February 2009

AbstractThe coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2′‐phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38–43). In order to analyze the structural changes involved, we have determined 4 high‐resolution crystal structures, i.e., the structures of the wild‐type enzyme (1.86 Å resolution, R‐factor of 16.8%), of the wild‐type enzyme ligated with NADP (2.0 Å, 20.8%), of the NAD‐dependent mutant (1.74 Å, 16.8%), and of the NAD‐dependent mutant ligated with NAD (2.2 Å, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD‐binding site assumes a less rigid structure than the NADP site of the wild‐type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 Å from the sites of the mutations, this observation reminds us that mutations may have important long‐range consequences that are difficult to anticipate.
Structure comparison of the pheromones Er‐1, Er‐10, and Er‐2 from Euplotes raikovi
Authors: Luginbühl Peter , Ottiger Marcel , Mronga Siggi , WÜthrich Kurt 
Published: February 2009

AbstractThe NMR structures of the homologous pheromones Er‐1, Er‐10, and Er‐2 from the ciliated protozoan Euplotes raikovi are compared. For all 3 proteins the molecular architecture is made up of an antiparallel 3‐helix bundle. The preservation of the core part of the structure is directly manifested by similar patterns of slowed backbone amide proton exchange rates, hydrogen bond formation, and relative solvent accessibility. To align the 6 half‐cystine residues in the individual sequences within the preserved 3‐dimensional core structure, several deletions and insertions had to be introduced that differ from those previously proposed on the basis of the primary structures. Of special interest is a deletion in the second helix of Er‐2, which is accommodated by a transition from an α‐helix in Er‐1 and Er‐10 to a 310‐helix in Er‐2. The most significant structural differences are located in the C‐terminal part of the proteins, which may have an important role in specific receptor recognition.
Identification of a molecular switch that selects between two crystal forms of bovine pancreatic trypsin inhibitor
Authors: Gallagher Warren H. , Croker Kevin M. 
Published: February 2009

AbstractTwo crystal forms of bovine pancreatic trypsin inhibitor are produced between pH 8.39 and 10.13 when crystals are grown at room temperature from solutions of 1.5 M potassium phosphate. Lower pH values favor the form II crystals, whereas higher pH values favor the form III. The transition from one crystal form to the other occurs at pH 9.35. We examined the crystal lattice contacts in both crystal forms and identified an unusual interaction we believe explains these observations. Spanning the crystallographic 2‐fold axis in form III crystals, the Lys 41 side‐chain amino nitrogens from 2 symmetry‐related molecules are only 2.72 Å apart, implying they are hydrogen bonded to one another. In form II crystals, the Lys 41 side‐chain amino group is protonated and forms a salt bridge with a solvent‐derived phosphate group. For the Lys 41 side‐chain amino groups to hydrogen bond in form III crystals, at least 1 member of the pair must be deprotonated. The transition that occurs at pH 9.35 marks the pKa for deprotonation. In solution, the pKa for the Lys 41 side chain is around 10.8. The pKa for one of the interacting Lys 41 side chains in form III crystals is therefore shifted downward by about 1.5 pH units. The energy for lowering the pKa value comes from the many additional inter‐molecular hydrogen bonds that are present in form III crystals: 19 compared to only 8 in form II crystals.