bluuebunny/crossref_metadata_2025 huggingface dataset:
Isoprenyl diphosphate synthases: Protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure
Authors: Chen Anjun , Dale Poulter C. , Kroon Paulos A.
Published: July 2010

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain‐elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate‐rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of α‐helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.
Matrix‐assisted laser desorption mass spectrometric peptide mapping of proteins separated by two‐dimensional gel electrophoresis: Determination of phosphorylation in synapsin I
Authors: Zhang Wenzhu , Czernik Andrew J. , Chait Brian T. , Yungwirth Tom , Aebersold Ruedi
Published: July 2010

A technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2‐dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys‐C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix‐assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (± 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2‐dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin‐dependent protein kinases, which appear to control their biological activity.
Charge reversal at the P3′ position in protein C optimally enhances thrombin affinity and activation rate
Authors: Richardson Mark A. , Gerlitz Bruce , Grinnell Brian W.
Published: July 2010

We have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3′ Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin‐catalyzed activation of D172N, D172G, and D172A was increased 4‐9‐fold compared to wildtype HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free a‐thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3′ Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3′ (D172K), there was a 30‐fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5‐fold) on the activation rate and affinity for free α‐thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium‐dependent and ‐independent effects of the acidic P3′ residue.
Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody
Authors: Schildbach Joel F. , Shaw Shyh‐Yu , Panka David J. , Near Richard I. , Bruccoleri Robert E. , Novotny Jiri , Sheriff Steven , Jeffrey Philip D. , Haber Edgar , Herzenberg Leonard A. , Jager Gina C. , Parks David R. , Margolies Michael N.
Published: July 2010

Two distinct spontaneous variants of the murine anti‐digoxin hybridoma 26‐10 were isolated by fluorescenceactivated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26‐10. Replacement of Asn with Gln reduced affinity for digoxin 10‐fold relative to the wild‐type antibody, but maintained wild‐type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90‐fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16‐acetylated haptens and haptens with a saturated lactone.

The X‐ray crystal structure of the 26‐10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acud Sci USA 90: 10310‐10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild‐type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild‐type and Gln‐mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild‐type hydrogen bonding, and molecular modeling suggests that the reduced side‐chain volume also provides space that can accommodate a congener with a 16‐acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.
Comparison of the structures and the crystal contacts of trypanosomal triosephosphate isomerase in four different crystal forms
Authors: Radhakishan K.V. , Zeelen Johan P.H. , Noble Martin E.M. , Borchert Torben V. , Wierenga Rik K.
Published: July 2010

Triosephosphate isomerase (TIM) is a dimeric enzyme consisting of 2 identical subunits. Trypanosomal TIM can be crystallized in 4 different spacegroups: P212121, C2(big cell), C2(small cell), and P1. The P1 crystal form only grows in the presence of 1.4 M DMSO; there are 2 DMSO binding sites per subunit. The structures have been refined at a resolution of 1.83 Å, 2.10 Å, 2.13 Å, and 1.80 Å, respectively. In the 4 different spacegroups the TIM subunit can be observed in the context of 7 different crystallographic environments. In the C2 cells, the dimer 2‐fold axis coincides with a crystallographic 2‐fold axis. The similarities and differences of the 7 subunits are discussed. In 6 subunits the flexible loop (loop 6) is open, whereas in the P212121 cell, the flexible loop of subunit 2 is in an almost closed conformation. The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.
Crystal structures of pheasant and guinea fowl egg‐white lysozymes
Authors: Lescar Julien , Souchon Hélene , Alzari Pedro M.
Published: July 2010

The crystal structures of pheasant and guinea fowl lysozymes have been determined by X‐ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6122 with cell dimensionsa= 89.2 Å andc= 61.7 Å. The structure was refined to a final crystallographicR‐factor of 17.0% for 8,854 observed reflections in the resolution range 6‐1.9 Å. Crystals of pheasant lysozyme are tetragonal, space group P43212, witha= 98.9 Å,c= 69.3 Å and 2 molecules in the asymmetric unit. The finalR‐factor is 17.8% to 2.1 Å resolution. The RMS deviation from ideality is 0.010 Å for bond lengths and 2.5° for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N‐terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3‐dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the Caatom of Gly 41 and absent in lysozymes from other species (which have a large side‐chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other C‐type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody‐bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen‐antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation inc‐type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes.
Thermodynamic characterization of an artificially designed amphiphilic α‐helical peptide containing periodic prolines: Observations of high thermal stability and cold denaturation
Authors: Kitakuni Eiichi , Kuroda Yutaka , Oobatake Motohisa , Tanaka Toshiki , Nakamura Haruki
Published: July 2010

To investigate the structural stability of proteins, we analyzed the thermodynamics of an artificially designed 30‐residue peptide. The designed peptide, NH2‐EELLPLAEALAPLLEALLPLAEALAPLLKK‐COOH (PERI COIL‐l), with prolines at i + 7 positions, forms a pentameric α‐helical structure in aqueous solution. The thermal denaturation curves of the CD at 222 nm (pH 7.5) show an unusual cold denaturation occurring well above 0 °C and no thermal denaturation is observable under 90 °C. This conformational change is reversible and depends on peptide concentration. A 2‐state model between the monomeric denatured state (5D) and the pentameric helical state (H5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL‐1 with concentrations between 23 and 286 μM. The analysis was carried out by a nonlinear least‐squares method using 3 fitting parameters: the midpoint temperature, Tm, the enthalpy change, ΔH(Tm), and the heat capacity change, Δ Cp. The association number (n= 5) was determined by sedimentation equilibrium and was not used as a fitting parameter. The heat capacity change suggests that the hydrophobic residues are buried in the helical state and exposed in the denatured one, as it occurs normally for natural globular proteins. On the other hand, the enthalpy and the entropy changes have values close to those found for coiled‐coils and are quite distinct from typical values reported for natural globular proteins. In particular, the enthalpy change extrapolated at 110 °C is about 3 kJ/mol per amino acid residue, i.e., half of the value found for globular proteins. Thus, the helices of PERI COIL‐1, observed by CD, would be stabilized by entropic effect rather than enthalpic effect. This might be a general feature for de novo designed proteins that lack the rigid tertiary structure, and are mainly stabilized by nonspecific hydrophobic interactions, as well as for some molten globules of natural proteins.
Helix propensities of the amino acids measured in alanine‐based peptides without helix‐stabilizing side‐chain interactions
Authors: Chakrabartty Avijit , Kortemme Tanja , Baldwin Robert L.
Published: July 2010

Helix propensities of the amino acids have been measured in alanine‐based peptides in the absence of helix‐stabilizing side‐chain interactions. Fifty‐eight peptides have been studied. A modified form of the Lifson‐Roig theory for the helix‐coil transition, which includes helix capping (Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL, 1994, Biochemistry 33:3396‐3403), was used to analyze the results. Substitutions were made at various positions of homologous helical peptides. Helix‐capping interactions were found to contribute to helix stability, even when the substitution site was not at the end of the peptide. Analysis of our data with the original Lifson‐Roig theory, which neglects capping effects, does not produce as good a fit to the experimental data as does analysis with the modified Lifson‐Roig theory. At 0 °C, Ala is a strong helix former, Leu and Arg are helix‐indifferent, and all other amino acids are helix breakers of varying severity. Because Ala has a small side chain that cannot interact significantly with other side chains, helix formation by Ala is stabilized predominantly by the backbone (“peptide H‐bonds”). The implication for protein folding is that formation of peptide H‐bonds can largely offset the unfavorable entropy change caused by fixing the peptide backbone. The helix propensities of most amino acids oppose folding; consequently, the majority of isolated helices derived from proteins are unstable, unless specific side‐chain interactions stabilize them.
Structural characteristics and stabilizing principles of bent β‐strands in protein tertiary architectures
Authors: Daffner Carola , Chelvanayagam Gareth , Argos Patrick
Published: July 2010

β‐Strands as constituents of β‐pleated sheets in protein tertiary structures often display considerable distortion from a purely extended conformation. The dislocation types are often characterized as “bulging,” “twisting,” and “bending.” The former 2 properties have been extensively studied and classified. In this work an investigation of bent β‐structures is undertaken. The structural characteristics examined included the bending angles within and out of the principal strand plane, their distribution among various strand types such as parallel and antiparallel, the amino acid preferences at bend sites, and the usage of charged and polar residues for stabilization through interactive anchoring with other atoms of the β‐sheet within which the bent strand lies.
Kringle‐kringle interactions in multimer kringle structures
Authors: Padmanabhan K. , Wu T.‐P. , Ravichandran K. G. , Tulinsky A.
Published: July 2010

The crystal structure of a monoclinic form of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the orthorhombic structure as a model and it has been refined by restrained least‐squares methods to an R factor of 16.4% at 2.25 Å resolution. The X‐PLOR structure of kringle 2 of tissue plasminogen activator (t‐PAK2) has been refined further using PROFFT (R = 14.5% at 2.38 Å resolution). The PGK4 structure has 2 and t‐PAK2 has 3 independent molecules in the asymmetric unit. There are 5 different noncrystallographic symmetry “dimers” in PGK4. Three make extensive kringle‐kringle interactions related by noncrystallographic 2, screw axes without blocking the lysine binding site. Such associations may occur in multikringle structures such as prothrombin, hepatocyte growth factor, plasminogen (PG), and apolipoprotein [a]. The t‐PAK2 structure also has noncrystallographic screw symmetry (31) and mimics fibrin binding mode by having lysine of one molecule interacting electrostatically with the lysine binding site of another kringle. This ligand‐like binding interaction may be important in kringle‐kringle interactions involving non‐lysine binding kringles with lysine or pseudo‐lysine binding sites. Electrostatic intermolecular interactions involving the lysine binding site are also found in the crystal structures of PGK1 and orthorhombic PGK4. Anions associate with the cationic centers of these and t‐PAK2 that appear to be more than occasional components of lysine binding site regions.
Receptor binding properties of four‐helix‐bundle growth factors deduced from electrostatic analysis
Authors: Demchuk Eugene , Mueller Thomas , Oschkinat Hartmut , Sebald Walter , Wade Rebecca C.
Published: July 2010

Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4‐α‐helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormone‐receptor recognition. In order to examine the specificity of hormone‐receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin‐2 (IL‐2), interleukin‐4 (IL‐4), granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and the hGH and IL‐4 receptors.

The binding surfaces of hGH and its receptor, and of IL‐4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2‐fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM‐CSF and IL‐2 lack such symmetry, consistent with their known high affinity for hetero‐oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero‐oligomeric model for a high‐affinity IL‐4 receptor and suggests a possible new receptor binding mode for G‐CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal‐transducing proteins and their receptors.
A comparison of X‐ray and NMR structures for human endothelin‐1
Authors: Wallace B.A. , Janes Robert W. , Bassolino Donna A. , Krystek Stanley R.
Published: July 2010

Direct comparisons between the recently solved X‐ray and NMR structures of human endothelin‐1 with respect to secondary structure, RMS deviations, surface accessibilities, and side‐chain conformers indicate important differences in conformation, especially in the C‐terminus, but also in the central loop region, that are important for defining the specificity of binding. These differences are larger than seen for other X‐ray and NMR structures that have been compared. Comparisons between the X‐ray structure and the NMR NOE constraints highlight the regions of flexibility and environment‐induced diversity in the endothelin structures.
A procedure for detecting structural domains in proteins
Authors: Swindells Mark B.
Published: July 2010

A procedure is described for detecting domains in proteins of known structure. The method is based on the intuitively simple idea that each domain should contain an identifiable hydrophobic core. By applying the algorithm described in the companion paper (Swindells MB, 1995, Protein Sci 4:93–102) to identify distinct cores in multi‐domain proteins, one can use this information to determine both the number and the location of the constituent domains. Tests have shown the procedure to be effective on a number of examples, even when the domains are discontinuous along the sequence. However, deficiencies also occur when hydrophobic cores from different domains continue through the interface region and join one another.
Identification of a eukaryotic‐like protein kinase gene in Archaebacteria
Authors: Smith Randall F. , King Katherine Y.
Published: July 2010

Primary sequence patterns based on known conserved sites in eukaryotic protein kinases were used to search for eukaryotic‐like protein kinase sequences in a six‐frame translation of the bacterial subsection of GenBank. This search identified a previously unrecognized eukaryotic‐like protein kinase gene in three related methanogenic archaebacteria, Methanococcus vannielii, M. voltae, and M. thermolithotrophicus. The proposed coding sequences are located in orthologous open reading frames (ORFs): ORF547, ORF294, and ORF114, respectively. The C‐terminus of the ORFs contains 9 of the 11 subdomains characteristically conserved within the eukaryotic protein kinase catalytic domain. The N‐terminus of the ORFs is similar to a putative glycoprotease in Pasteurella haemolytica and its homologue in Escherichia coli, the orfX gene. This is the first report of a eukaryotic‐like protein kinase sequence observed in Archaebacteria.
Identification of the Serratia endonuclease dimer: Structural basis and implications for catalysis
Authors: Miller Mitchell D. , Krause Kurt L.
Published: July 2010

The Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single‐ and double‐stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X‐ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461–468). We now confirm the dimeric nature of this enzyme through light‐scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy‐terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 Å× ± 35 Å × 90 Å. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual “cubic” water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.
A model for the regulation of D‐3‐phosphoglycerate dehydrogenase, a Vmax‐type allosteric enzyme
Authors: Grant Gregory A. , Schuller David J. , Banaszak Leonard J.
Published: July 2010

Escherichia coli D‐3‐phosphoglycerate dehydrogenase (ePGDH) is a tetramer of identical subunits that is alloste‐rically inhibited by L‐serine, the end product of its metabolic pathway. Because serine binding affects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the Vmax type. Inhibition by a variety of amino acids and analogues of L‐serine indicate that all three functional groups of serine are required for optimal interaction. Removing or altering any one functional group results in an increase in inhibitory concentration from micromolar to millimolar, and removal or alteration of any two functional groups removes all inhibitory ability. Kinetic studies indicate at least two serine‐binding sites, but the crystal structure solved in the presence of bound serine and direct serine‐binding studies show that there are a total of four serine‐binding sites on the enzyme. However, approximately 85% inhibition is attained when only two sites are occupied. The three‐dimensional structure of ePGDH shows that the serine‐binding sites reside at the interface between regulatory domains of adjacent subunits. Two serine molecules bind at each of the two regulatory domain interfaces in the enzyme. When all four of the serines are bound, 100% inhibition of activity is seen. However, because the domain contacts are symmetrical, the binding of only one serine at each interface is sufficient to produce approximately 85% inhibition. The tethering of the regulatory domains to each other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reorientation that must accompany a catalytic cycle. It is suggested that part of the conformational change may involve a hinge formed in the vicinity of the union of two antiparallel β‐sheets in the regulatory domains. The tethering function of serine, in turn, appears to prevent the substrate‐binding domain from closing the cleft between it and the nucleotide‐binding domain, which may be necessary to form a productive hydrophobic environment for hydride transfer. Thus, the structure provides a plausible model that is consistent with the binding and inhibition data and that suggests that catalysis and inhibition in this rare Vmax‐type allosteric enzyme is based on the movement of rigid domains about flexible hinges.
The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 Å resolution
Authors: Zhang Yihong , Bond Charles S. , Bailey Susan , Cunningham Mark L. , Fairlamb Alan H. , Hunter William N.
Published: July 2010

Trypanothione reductase (TR) is an NADPH‐dependent flavoprotein unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved trypanocidal drugs. We present details of the structure of TR from the human pathogen Trypanosoma cruzi, the agent responsible for Chagas' disease or South American trypanosomiasis. The structure has been solved by molecular replacement, using as the starting model the structure of the enzyme from the nonpathogenic Crithidia fasciculata, and refined to an R‐factor of 18.9% for 53,868 reflections with F κ σF between 8.0 and 2.3 Å resolution. The model comprises two subunits (968 residues), two FAD prosthetic groups, two maleate ions, and 419 water molecules. The accuracy and geometry of the enzyme model is improved with respect to the C. fasciculata enzyme model. The new structure is described and specific features of the enzyme involved in substrate interactions are compared with previous models of TR and related glutathione reductases from human and Escherichia coli. Structural differences at the edge of the active sites suggest an explanation for the differing specificities toward glutathionylspermidine disulfide.
A recombinant peptide model of the first nucleotide‐binding fold of the cystic fibrosis transmembrane conductance regulator: Comparison of wild‐type and ΔF508 mutant forms
Authors: Yike Iwona , Ye Jin , Zhang Yan , Manavalan Parthasarathy , Gerken Thomas A. , Dearborn Dorr G.
Published: July 2010

A series of recombinant peptides, each including the sequence proposed to be the first nucleotide‐binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native‐like properties. The peptide NBD1F, which contains the 267‐amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine‐HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol‐ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP‐γS is 1.0 ± 0.4 μM and for ADP 8.8 ± 3.1 μM. The native‐like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5′‐adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5′‐adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBD1F. The CD spectra of NBD1F and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% α‐helix and 23% β‐sheet, with insignificant differences in the amounts of β‐turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide‐binding domain of CFTR.
Contact interactions method: A new algorithm for protein folding simulations
Authors: Toma Lucio , Toma Salvatore
Published: July 2010

Computer simulations of simple exact lattice models are an aid in the study of protein folding process; they have sometimes resulted in predictions experimentally proved. The contact interactions (CI) method is here proposed as a new algorithm for the conformational search in the low‐energy regions of protein chains modeled as copolymers of hydrophobic and polar monomers configured as self‐avoiding walks on square or cubic lattices. It may be regarded as an extension of the standard Monte Carlo method improved by the concept of cooperativity deriving from nonlocal contact interactions. A major difference with respect to other algorithms is that criteria for the acceptance of new conformations generated during the simulations are not based on the energy of the entire molecule, but cooling factors associated with each residue define regions of the model protein with higher or lower mobility. Nine sequences of length ranging from 20 to 64 residues were used on the square lattice and 15 sequences of length ranging from 46 to 136 residues were used on the cubic lattice. The CI algorithm proved very efficient both in two and three dimensions, and allowed us to localize energy minima not localized by other searching algorithms described in the literature. Use of this algorithm is not limited to the conformational search, because it allows the exploration of thermodynamic and kinetic behavior of model protein chains.
Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli
Authors: Qamar Seema , Marsh Katherine , Berry Alan
Published: July 2010

Treatment of the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli with the arginine‐specific α‐dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6‐bisphosphate, or by inorganic phosphate. Modification with [7‐14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP‐aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg‐331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady‐state kinetic parameters revealed that mutation of Arg‐331 dramatically increased the Km for fructose 1,6‐bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2‐phosphoglycolate, were found between the wild‐type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3‐phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase‐substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the Km for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6‐bisphosphate was dramatically increased. Taken together, these results show that Arg‐331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C‐6 phosphate group of the substrate.
Extending the C2 domain family: C2s in PKCs δ, ϵ,η,θ, phospholipases, GAPs, and perforin
Authors: Pointing C.P. , Parker P.J.
Published: July 2010

Various membrane lipid metabolites, generated by phospholipases C and D (PLCs, PLDs), are known to regulate the activities of protein kinases C (PKCs) and GTP‐ase activating proteins (GAPs) in a range of cellular processes. Conventional Ca2+‐dependent PKCs (α, βI, βII, and γ), PLCs, and various GAPs are all known to contain copies of a phospholipid‐binding domain, termed C2 or CalB. Here we recognize that C2 domains are also present in “new” Ca2+ ‐independent PKCs (δ, η, η, and θ), other kinases, a eukaryotic PLD, the breakpoint cluster region (BCR) gene product, and two further GAPS. Twenty‐two previously unrecognized C2 domain sequences are presented, which include a single copy in the mammalian poreforming proteins, perforin.
An alternative topological model for Escherichia coli OmpA
Authors: Stathopoulos Christos
Published: July 2010

The current topological model for the Escherichia coli outer membrane protein OmpA predicts eight N‐terminal transmembrane segments followed by a long periplasmic tail. Several recent reports have raised serious doubts about the accuracy of this prediction. An alternative OmpA model has been constructed using (1) computer‐aided predictions developed specifically to predict topology of bacterial outer membrane porins, (2) the results of two reports that identified sequence homologies between OmpA and other peptidoglycan‐associated proteins, and (3) biochemical, immunochemical, and genetic topological data on proteins of the OmpA family provided by numerous previous studies. The new model not only agrees with the varied experimental data concerning OmpA but also provides an improved understanding of the relationship between the structure and the multifunctional role of OmpA in the bacterial outer membrane.
Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy
Authors: Gronenborn Angela M. , Clore G. Marius
Published: July 2010

simple and rapid method based on 15N labeling and 1H‐15N heteronuclear single quantum coherence spectroscopy is presented to directly assess the structural integrity of overexpressed proteins in crude Escherichia coli extracts without the need for any purification. The method is demonstrated using two different expression systems and two different proteins, the B1 immunoglobulin‐binding domain of streptococcal protein G (56 residues) and human interleukin‐1β (153 residues). It is shown that high quality 1H‐15N correlation spectra, recorded in as little as 15 min and displaying only cross‐peaks arising from the overexpressed protein of interest, can be obtained from crude E. coli extracts.
Structural resiliency of an EGF‐like subdomain bound to its target protein, thrombin
Authors: Hrabal Richard , Ni Feng , Komives Elizabeth A.
Published: July 2010

The thrombin‐bound structures of native peptide fragments from the fifth EGF‐like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF‐like structure of an antiparallel β‐sheet, a novel structural motif observed for a bound peptide in protein‐peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF‐like domains, are unfavorably exposed in the complex of thrombin with the EGF‐like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique β‐sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side‐chain interactions within a specific environment may dictate the folding of bound peptides in protein‐peptide complexes.
Are turns required for the folding of ribonuclease T1?
Authors: Garrett James B. , Mullins Leisha S. , Raushel Frank M.
Published: July 2010

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys‐2 and Cys‐10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino‐terminus and carboxyl‐terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent‐exposed loops at Ser‐35 (cp35Sl), Asp‐49 (cp49D1), Gly‐70 (cp70G1), and Ser‐96 (cp96S1). These circularly permuted RNase T1 mutants retained 35–100% of the original catalytic activity for the hydrolysis of guanylyl(3′ → 5′)cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild‐type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild‐type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight β‐turn between Glu‐82 and Gln‐85. New termini were inserted at Asn‐83 (cp83N1), Asn‐84 (cp84N1), and Gln‐85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.
Second‐site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase
Authors: Chen Ridong , Dean Antony M. , Grobler Jay A. , Hurley James H.
Published: July 2010

Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged γ‐carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/Ki‐isocitrat/KmNADP) by a factor of 107. Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second‐site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D‐helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 105 higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged γ‐carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild‐type asparagine. Repacking around the leucine forces the amino‐terminus of the D‐helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the γ‐carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.
Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments
Authors: Gross Martin , Wallimann Theo , Wyss Markus , Furter‐Graves Elizabeth M. , Furter Rolf
Published: July 2010

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease‐sensitive site in M‐CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi‐CK) into two protein fragments encompassing amino acids [1–167] and [168–380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two‐step procedure. In contrast, co‐expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi‐CK by far‐ and near‐UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C‐terminal fragment refolded into a native‐like conformation in vitro, whereas the N‐terminal fragment was largely unfolded. Prefolded [168–380] interacted in vitro with [1–167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1, = 17 μM) and then isomerize slowly to an active enzyme (k2 = 0.12 min−1; k‐2 = 0.03 min−1). Our data suggest that the C‐terminal fragment of Mi‐CK represents an autonomous folding unit, and that the folding of the C‐terminal part might precede the conformational stabilization of the N‐terminal moiety in vivo.
Computational method for relative binding energies of enzyme‐substrate complexes
Authors: Zhang Tao , Koshland Daniel E.
Published: July 2010

A computational method for estimating the relative binding free energies of enzyme‐substrate complexes is described that combines electrostatic and solvation models and X‐ray crystallographic data. The polar contribution is evaluated by the Poisson‐Boltzman equation. The nonpolar contribution is evaluated by solvent transfer data and surface area calculations. This algorithm was used to calculate the relative binding energies of 63 pairs of nine different mutant proteins with seven different substituted R‐malate substrates of Escherichia coli isocitrate dehydrogenase. Comparison of calculated values with the experimentally observed values shows a high degree of correlation.
On the role of surface tension in the stabilization of globular proteins
Authors: Lin Tiao‐Yin , Timasheff Serge N.
Published: July 2010

The stabilization of proteins by a variety of co‐solvents can be related to their property of increasing the surface tension of water. It is demonstrated that, during the thermal unfolding of proteins, this increase of the surface tension can be overcome by the increase in the temperature of the solution at the midpoint of the transition, Tm, and the weak binding of co‐solvent molecules. Three such co‐solvents were studied: trehalose, lysine hydrochloride (LysHCl), and arginine hydrochloride (ArgHCl). Trehalose and LysHCl increase the midpoint of Tm. The increase of the surface tension by addition of trehalose is completely compensated by its decrease due to the increase in Tm. However, for LysHCl, the increase of the surface tension by the co‐solvent is partly reduced by its binding to the protein. For trehalose, preferential interaction measurements with RNaseA demonstrate that it is totally excluded from the protein. In contrast, LysHCl gives evidence of binding to RNaseA. ArgHCl also increases the surface tension of water. Nevertheless, Tm of RNaseA decreases on addition of ArgHCl to the solution. Preferential interaction measurements showed very small values of preferential hydration of the native protein, indicating extensive binding of ArgHCl to the protein. During unfolding, the amount of additional ArgHCl binding is sufficiently large to counteract the surface tension effects, and the protein is destabilized. Therefore, although surface tension appears to be a critical factor in the stabilization of proteins, its increase by co‐solvent does not ensure increased stabilization. The binding of ligands can reduce significantly, or even overwhelm, its effects.
Woodward's reagent K inactivation of Escherichia coli L‐threonine dehydrogenase: Increased absorbance at 340–350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups
Authors: Johnson Adam R. , Dekker Eugene E.
Published: July 2010

L‐Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L‐threonine (60 mM) is ∼50% as effective. Tryptic digestion of WRK‐modified TDH followed by HPLC fractionation (pH 6.2) yields four 340‐nm‐absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35–43), whereas peptide II is TAICGTDVHIY (residues 35–45). Peptides not protected are TMLDTMNHGGR (III, residues 248–258) and NCRGGRTHLCR (IV, residues 98–108). Absorbance spectra of these WRK‐peptides were compared with WRK adducts of imidazole, 2‐hydroxy‐ethanethiolate, and acetate. Peptides III and IV have pH‐dependent λmax values (340–350 nm), consistent with histidine modification. Peptide I has a pH‐independent λmax (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys‐38 in the active site of TDH; modification of His‐105 and His‐255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340–350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.
pH‐Dependent self‐association of the Src homology 2 (SH2) domain of the Src homologous and collagen‐like (SHC) protein
Authors: Réty Stéphane , Fütterer Klaus , Grucza Richard A. , Munoz Consuelo M. , Frazier William A. , Waksman Gabriel
Published: July 2010

The Src homologous and collagen‐like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 Å. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide‐binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C‐terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry‐related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH‐dependent manner where low pH conditions (∼pH 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.