bluuebunny/crossref_metadata_2025_split huggingface dataset:
Irreversible thermal denaturation ofTorpedo californicaacetylcholinesterase
Authors: Kreimer David I. , Shnyrov Valery L. , Villar Enrique , Silman Israel , Weiner Lev 
Published: February 2009

AbstractThermal denaturation ofTorpedo californicaacetylcholinesterase, a disulfide‐linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two‐state kinetic scheme N → D, with activation energy 131 ± 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 ± 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol‐specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.
Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site
Authors: Liaw Shwu‐Huey , Kuo Ichun , Eisenberg David 
Published: February 2009

AbstractGlutamine synthetase (GS) catalyzes the ATP‐dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel‐shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X‐ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50′, Ser 53′ of the adjacent sub‐unit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the γ‐glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 Å, between n1 and Tl+ is 4 Å, and between n2 and Tl+ is 7 Å. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the “side‐to‐side” intersubunit interaction through the cation‐protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50′ toward this monovalent cation site, essentially forming the site. This observation supports a two‐step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form γ‐glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the γ‐glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate γ‐glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.
Significance of structural changes in proteins: Expected errors in refined protein structures
Authors: Stroud Robert M. , Fauman Eric B. 
Published: February 2009

AbstractA quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by “machine learning” principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal B factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six‐parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.
How to measure and predict the molar absorption coefficient of a protein
Authors: Pace C. Nick , Vajdos Felix , Fee Lanette , Grimsley Gerald , Gray Theronica 
Published: February 2009

AbstractThe molar absorption coefficient, ε, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring ε for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319–326] and is based on data from Edelhoch [1967, Biochemistry 6:1948–1954].) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average ε values for these chromophores in a sample of 18 well‐characterized proteins have been estimated, and the ε values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average ε values for the proteins are less than the ε values measured in any of the solvents. For Tyr, the average ε values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured ε values for 80 proteins, the ε at 280 nm of a folded protein in water, ε(280), can best be predicted with this equationϵ(280) (M−1 cm−1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125).These ε(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict ε.
New protein functions in yeast chromosome VIII
Authors: Ouzounis Christos , Bork Peer , Casari Georg , Sander Chris 
Published: February 2009

AbstractThe analysis of the 269 open reading frames of yeast chromosome VIII by computational methods has yielded 24 new significant sequence similarities to proteins of known function. The resulting predicted functions include three particularly interesting cases of translation‐associated proteins: peptidyl‐tRNA hydrolase, a ribosome recycling factor homologue, and a protein similar to cytochrome b translational activator CBS2. The methodological limits of the meaningful transfer of functional information between distant homologues are discussed.
Structural similarities in the noncatalytic domains of phenylalanyl‐tRNA and biotin synthetases
Authors: Safro Mark , Mosyak Lidia 
Published: February 2009

AbstractDetailed comparison between the structures of the Escherichia coli biotin synthetase/repressor protein (BirA) and the recently solved Thermus thermophilus phenylalanyl‐tRNA synthetase (PheRS) reveals significant similarities outside their respective catalytic domains. These comprise a DNA‐binding α+β domain and an Src‐homology 3 (SH3)‐like domain that were observed in both enzymes. This similarity provides a novel example in which all domains of one multidomain protein appear to be constituents of the other multidomain protein and supports a concept of a common ancestor for two different synthetase families.
Identification of the catalytic histidine residue participating in the charge‐relay system of carboxypeptidase Y
Authors: Jung Giman , Ueno Hiroshi , Hayashi Rikimaru , Liao Ta‐Hsiu 
Published: February 2009

AbstractThe essential histidine residue of carboxypeptidase Y (CPY) was modified by a site‐specific reagent, a chloromethyl‐ketone derivative of benzyloxycarbonyl‐L‐phenylalanine. The single modified histidine residue was converted to Nτ ‐carboxymethyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic‐thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val‐Phe‐Asp‐Gly‐Gly‐cmHis‐MetO2‐Val‐Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X‐ray analysis to participate in the charge‐relay system of CPY.
Crystallization and preliminary X‐ray diffraction analysis of recombinant pentalenene synthase
Authors: Lesburg Charles A. , Lloyd Matthew D. , Cane David E. , Christianson David W. 
Published: February 2009

AbstractRecombinant pentalenene synthase, a 42.5‐kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P63 with unit cell dimensions a = b = 183.5 Å and c = 56.5 Å. Hexagonal prismatic crystals, approximately 0.2 × 0.2 × 0.3 mm, diffract to approximately 2.9 Å resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five‐membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.
3D domain swapping: A mechanism for oligomer assembly
Authors: Bennett Melanie J. , Schlunegger Michael P. , Eisenberg David 
Published: February 2009

Abstract3D domain swapping is a mechanism for forming oligomeric proteins from their monomers. In 3D domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical protein chain. The result is an intertwined dimer or higher oligomer, with one domain of each subunit replaced by the identical domain from another subunit. The swapped “domain” can be as large as an entire tertiary globular domain, or as small as an α‐helix or a strand of a β‐sheet. Examples of 3D domain swapping are reviewed that suggest domain swapping can serve as a mechanism for functional interconversion between monomers and oligomers, and that domain swapping may serve as a mechanism for evolution of some oligomeric proteins. Domain‐swapped proteins present examples of a single protein chain folding into two distinct structures.
Phosphorylation of serine‐46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure
Authors: Pullen Katherine , Rajagopal Ponni , Klevit Rachel E. , Branchini Bruce R. , Reizer Jonathan , Saier Milton H. , Scholtz J. Martin , Huffine Mary Elizabeth 
Published: February 2009

AbstractThe serine‐phosphorylated form of histidine‐containing protein (HPr), a component of the phosphoenol‐pyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies. The results indicate that phosphorylation of Ser 46, the N‐cap of α‐helix‐B, does not cause a conformational change but rather stabilizes the helix. Amide proton exchange rates in helix‐B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a ΔΔG of 0.7‐0.8 kcal mol−1. A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.
Comparison of atomic solvation parametric sets: Applicability and limitations in protein folding and binding
Authors: Juffer André H. , Eisenhaber Frank , Hubbard Simon J. , Walther Dirk , Argos Patrick 
Published: February 2009

AbstractAtomic solvation parameters (ASP) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein‐ligand complexes. They are also included in several molecular mechanics computer programs. In this work, a total of eight atomic solvation parametric sets has been employed to calculate the solvation contribution to the free energy of folding ΔGs for 17 proteins. A linear correlation between ΔGs and the number of residues in each protein was found for each ASP set. The calculations also revealed a great variety in the absolute value and in the sign of ΔGs values such that certain ASP sets predicted the unfolded state to be more stable than the folded, whereas others yield precisely the opposite. Further, the solvation contribution to the free energy of association of helix pairs and to the disassociation of loops (connection between secondary structural elements in proteins) from the protein tertiary structures were computed for each of the eight ASP sets and discrepancies were evident among them.
A simple and fast approach to prediction of protein secondary structure from multiply aligned sequences with accuracy above 70%
Authors: Mehta Perdeep K. , Heringa Jaap , Argos Patrick 
Published: February 2009

AbstractTo improve secondary structure predictions in protein sequences, the information residing in multiple sequence alignments of substituted but structurally related proteins is exploited. A database comprised of 70 protein families and a total of 2, 500 sequences, some of which were aligned by tertiary structural superpositions, was used to calculate residue exchange weight matrices within α‐helical, β‐strand, and coil substructures, respectively. Secondary structure predictions were made based on the observed residue substitutions in local regions of the multiple alignments and the largest possible associated exchange weights in each of the three matrix types. Comparison of the observed and predicted secondary structure on a per‐residue basis yielded a mean accuracy of 72.2%. Individual α‐helix, β‐strand, and coil states were respectively predicted at 66.4, 66.7, and 75.8°7o correctness, representing a well‐balanced three‐state prediction. The accuracy level, verified by cross‐validation through jack‐knife tests on all protein families, dropped, on average, to only 70.9%, indicating the rigor of the prediction procedure. On the basis of robustness, conceptual clarity, accuracy, and executable efficiency, the method has considerable advantage, especially with its sole reliance on amino acid substitutions within structurally related proteins.
Spectroscopic characterization of rhino viral protease 2a: Zn is essential for the structural integrity
Authors: Voss Tilman , Meyer Rainer , Sommergruber Wolfgang 
Published: February 2009

AbstractRecently, protease 2A of human rhinovirus 2 (HRV2 2A) was shown to require a zinc ion for the formation of an active enzyme although zinc is not involved mechanistically. The data presented clearly show that the zinc ion bound to a picornaviral‐specific motif represents an essential component of the native structure, probably representing a new Zn‐binding motif. This structure, containing mostly β‐strand elements as shown by CD spectroscopy, changes drastically upon removal of zinc. The zinc‐depleted form does represent an intermediate with mostly unchanged secondary structure, but not a fully denatured random coil as obtained by guanidinium hydrochloride. This is indicated by the blue‐shifted fluorescence spectra and by CD. The native protein exhibited a cooperative phase transition at 53 °C. In contrast, the zinc‐depleted form did not show any transition at all, again demonstrating the stabilizing role of the zinc ion. A structural intermediate was observed during thermal and pH denaturation that may represent a molten globule, as suggested by its ANS binding.
Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes
Authors: Heyse Stephan , Vogel Horst , Sänger Michael , Sigrist Hans 
Published: February 2009

AbstractA new method is presented for measuring sensitively the interactions between ligands and their membrane‐bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass‐like surface. In a first step, phospholipids carrying head‐group thiols were covalently immobilized onto SiO2‐TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N‐succinimidyl‐3‐maleimidopropionate, followed by the formation of thioethers between the surface‐grafted maleimides and the synthetic thiolipids. The surface‐attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self‐assembly from mixed lipid/detergent micelles. The step‐by‐step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50‐60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45‐50 Å, which corresponds to the thickness of a fluid bilayer membrane.Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid‐anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopeptide concentration in the outer monolayer.
The effects of filtration on protein nucleation in different growth media
Authors: Hirschler Joachim , Charon Marie‐Hélène , Fontecilla‐Camps Juan Carlos 
Published: February 2009

AbstractFiltration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease depended on the membrane cut‐off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 μm in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results.The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time‐dependent change in the size or the number of crystals per drop.Repeating the filtration experiments in agarose‐gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time‐dependent effect.
Catalytic ability and stability of two recombinant mutants of D‐amino acid transaminase involved in coenzyme binding
Authors: Van Ophem Peter W. , Pospischil Maria A. , Manning James M. , Ringe Dagmar , Peisach Daniel , Petsko Gregory , Soda Kenji 
Published: February 2009

AbstractOf the major amino acid side chains that anchor pyridoxal 5′‐phosphate at the coenzyme binding site of bacterial D‐amino acid transaminase, two have been substituted using site‐directed mutagenesis. Thus, Ser‐180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr‐31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild‐type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5‐15% lower concentration of urea or guanidine than did the wild‐type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5′‐phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild‐type and the S180A enzymes in the presence of normal D‐amino acid substrates. However, β‐chloro‐D‐alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild‐type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5‐phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild‐type enzymes because racemase activity, i.e., conversion of L‐alanine to D‐alanine, was higher than for the wild‐type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.
Nuclear magnetic resonance characterization of the N‐terminal thioredoxin‐like domain of protein disulfide isomerase
Authors: Kemmink Johan , Darby Nigel J. , Creighton Thomas E. , Dijkstra Klaas , Scheek Ruud M. 
Published: February 2009

AbstractA genetically engineered protein consisting of the 120 residues at the N‐terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods. The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and β‐sheet topology. The results confirm that PDI is a modular, multidomain protein. The last 20 residues of the N‐terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold. This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains.