bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural features of ribonucleotide reductase
Authors: Nikas Ioannis , McLauchlan John , Davison Andrew J. , Taylor William R. , Clements J. Barklie
Published: May 2005

Herpes simplex virus type 1 (HSV‐1) encodes a ribonucleotide reductase which comprises two polypeptides with sizes of 136,000 (RR1) and 38,000 mol. wt. (RR2). We have determined the entire DNA sequence specifying HSV‐1 RR1 and have identified two adjacent open reading frames in varicella‐zoster virus (VZV) which have homology to HSV RR1 and RR2; the predicted sizes for the VZV RR1 and RR2 polypeptides are 87,000 and 35,000 mol. wt. respectively. Amino acid comparisons with RR1 and RR2 polypeptides from other organisms indicate that HSV‐1 RR1 contains a unique N‐terminal domain which is absent from other RR1 polypeptides apart from HSV‐2 RR1. These N‐terminal amino acid sequences are poorly conserved between HSV‐1 and HSV‐2 in contrast to the remainder of the protein which shows greater than 90% homology. Polypeptide structural predictions suggest that the HSV‐1 N‐terminal domain may be separated into two regions, namely, a β‐sheet structure followed by a nonstructured area. Across the remainder of RR1 and RR2, comparisons also reveal blocks of amino acids conserved between the different ribonucleotide reductases, and these may be important for enzyme activity. From predictions on the structure of these conserved blocks, we have proposed that the location of a substrate binding site within RR1 is centered on three conserved glycine residues in a region which is predicted to adopt a β‐sheet/turn/α‐helical structure;this approximates to the structure for ADP nucleotide binding folds. Finally, we propose that the promoters for the HSV and Eptein‐Barr virus (EBV) RR2 transcripts have evolved by separate evolutionary routes.
Detection of human somatic cell structural gene mutations by two‐dimensional electrophoresis
Authors: Hanash S.M. , Chu E.H.Y. , Kuick R. , Skolnick M. , Neel J. , Strahler J. , Pivirotto S. , Niezgoda W.
Published: May 2005

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two‐dimensional approach for mutagenesis studies at the protein level.
Structural analysis of purified beta‐adrenergic receptors
Authors: Fraser Claire M. , Kerlavage Anthony R. , Mariani Andrew P. , Venter J. Craig
Published: May 2005

We have characterized the structure of purified beta‐adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)‐tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta‐adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]‐iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta‐receptors were subjected to complete trypsin digestion and subsequent reverse‐phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta‐receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod‐shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta‐receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta‐adrenergic receptor and its quaternary structure.
Distribution of accessible surfaces of amino acids in globular proteins
Authors: Lawrence Charles , Auger Ivan , Mannella Carmen
Published: May 2005

Globular proteins fold into compact particles with interior amino acid residues shielded from the surounding aqueous environment. An early hypothesis holds that entropic hydophobic forces dirve this phenomenon. However, previous analyses based on a binary description of the accesible surfaces of amino acid residues in proteins did not support theis hypothesis. This report shows that a complete description of accessible surface areas is given by parametric distribution fuctions with three modes. The modes are formed by partitioning the available accessible surface area of the amino acids into three segments; the data for each segment are characterized by a mode‐specific model. In the “repulsive” mode, probabilities of accessibility decrease exponentially with exposed surface area, as predicted by the hydrophobic hypothesis. A distinct “buried” mode is needed to account for an excess of residues at or near zero accessibility for most amino acids, consistent with the use of binary descriptions of accessibility. A third mode exists which is termed “near neutral” because it is described by a nearly uniform distribution of accessibility for the hydrophiliic amino acids. Empirical energies calculated for the repulsive mode correlate well with measured free energies of transfer of amino acids from water to organic solvents, while those from the buried mode correlate well with measured free energies of hydration of the side chains. Poor cross correlations between these energies give an explanation for the previous conflict in interpreting these data.
Structure of an insect virus at 3.0 Å resolution
Authors: Hosur M.V. , Schmidt T. , Tucker R.C. , Johnson J.E. , Gallagher T.M. , Selling B.H. , Rueckert R.R.
Published: May 2005

We report the first atomic resolution structure of an insect virus determined by single crystal X‐ray diffraction. Black beetle virus has a bipartite RNA genome encapsulated in a single particle. The capsid contains 180 protomers arranged on a T = 3 surface lattice. The quaternary organization of the protomers is similar to that observed in the T = 3 plant virus structures. The protomers consist of a basic, crystallographically disordered amino terminus (64 residues), a β‐barrel as seen in other animal and plant virus subunits, an outer protrusion composed predominantly of β‐sheet and formed by three large insertions between strands of the barrel, and a carboxy terminal domain composed of two distorted helices lying inside the shell. The outer surfaces of quasi‐threefold related protomers form trigonal pyramidyl protrusions. A cleavage site, located 44 residues from the carboxy terminus, lies within the central cavity of the protein shell.

The structural motif observed in BBV (a shell composed of 180 eight‐stranded antiparallel β‐barrels) is common to all nonstatellite spherical viruses whose structures have so far been solved. This highly conserved shell architecture suggests a common origin for the coat protein of spherical viruses, while the primitive genome structure of BBV suggests that this insect virus represents an early stage in the evolution of spherical viruses from cellular genes.
Anisotropy and anharmonicity of atomic fluctuations in proteins: Analysis of a molecular dynamics simulation
Authors: Ichiye Toshiko , Karplus Martin
Published: May 2005

Positional probability density functions (pdf) for the atomic fluctuations are determined from a molecular dynamics simulation for hen egg‐white lysozyme. Most atoms are found to have motions that are highly anisotropic but only slightly anharmonic. The largest deviations from harmonic motion are in the direction of the largest rms fluctuations in the local principal axis frame. Backbone atoms tend to be more nearly harmonic than sidechain atoms. The atoms with the largest anharmonicities tend to have pdfs with multiple peaks, each of which is close to harmonic. Several model pdfs are evaluated on the basis of how well they fit probability densities from the dynamics simulations when parameterized in terms of the moments of the distribution. Gram‐Charlier and Edgeworth perturbation expansions, which have been successful in describing the motions of small molecules in crystals, are shown to be inadequate for the distributions found in the dynamics of proteins. Multipeaked distribution functions are found to be more appropriate.
Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family
Authors: Kimura Shioko , Ikeda‐Saito Masao
Published: May 2005

Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396–418 in thyroid peroxidase and 405–427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.
Peptide synthesis catalyzed by polyethylene glycol‐modified chymotrypsin in organic solvents
Authors: Gaertner Hubert F. , Puigserver Antoine J.
Published: May 2005

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene‐soluble modified enzyme readily catalyzed both aminolysis of N‐benzoyl‐L‐tyrosine p‐nitroanilide and synthesis of N‐benzoyl‐L‐tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L‐ as well as D‐amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives

The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl‐enzyme with subsequent synthesis of a peptide bond.
Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A
Authors: Brandhuber Barbara J. , Allured Viloya S. , Falbel Tanya G. , McKay David B.
Published: May 2005

Pseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP‐ribosyl, which catalyze the covalent transfer of an ADP‐ribose moiety of NAD+ to a target substrate. Availability of the three‐dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD+ binding site of exotoxin A: the binding of NAD+ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP‐ribosyl transferase activity.
Crystallization and preliminary X‐ray diffraction study of a protein with a high potential rubredoxin center and a hemerythrin‐type Fe center
Authors: Sieker L. C. , Turley S. , Prickril B. C. , LeGall J.
Published: May 2005

A newly discovered iron‐containing protein, isolated from the bacterium Desulfovibrio vulgaris (Hildenborough, NCIB 8303), has been crystallized. The molecule appears to be a dimer of mass 44kDa. This protein has iron centers with spectroscopic similarities to those in rubredoxins and in hemerythrins.

The X‐ray diffraction shows symmetry consistent with space group I222 or I212121. Cell parameters are a = 49.2 Å, b = 81.3 Å, c= 100.1 Å, and α, β, γ = 90°. X‐ray diffraction data have been collected to 3.0 Å, and a search for useful heavy atom derivatives is in progress for the analysis of the crystal structure of this Fe‐protein.
Mechanism of protein folding: I. General considerations and refolding of myoglobin
Authors: Saitô Nobuhiko , Shigaki Takao , Kobayashi Yutaka , Yamamoto Masahiko
Published: May 2005

To explain the rapidity of the process of protein folding, we cite two aspects of hydrophobic interaction: its long‐range nature and the specificity of pairing after the formation of secondary structures. These two factors, when incorporated with the growth‐type mechanism, can determine the folding pathway of proteins. This mechanism is applied to myoglobin. Appropriate introduction of side chins of amino acid residues and the heme group attached to His 93 yield a refolded tertiary structure that is in good agreement with the native structure.
Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine‐185 protonates and deprotonantes during the photocycle
Authors: Braiman Mark S. , Mogi Tatsushi , Stern Lawrence J. , Hackett Neil R. , Chao Betty H. , Khorana H. Gobind , Rothschild Kenneth J.
Published: May 2005

The techniques of FTIR difference spectroscopy and site‐directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton‐pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr→Phe substitution, difference spectra have been obtained for the bR→K and bR→M photoreactions. Only the Tyr‐185→Phe mutation results in the disappearance of a set of bands that were previously shown to be due to protonation of a tryosinate during the br→K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United states of America 83:347, (1986)]. The Tyr‐185→Phe mutation also eliminates a set of bands in the bR→M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272‐cm−1 peak) are completely unaffected by the other ten Tyr→Phe mutations. Thus, tyrosinate‐185 gains a proton during the bR→K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr‐185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative CNH+ stretch band shifts from 1640 cm−1 in the wild type to 1636 cm−1 in the Tyr‐185→Phe mutant. A model that is consistent with these results is that Tyr‐185 is normally ionized and serves as a counter‐ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr‐185, which raises the pKa of the latter group and results in its protonation.
The aggregation state of spin‐labeled melittin in solution and bound to phospholipid membranes: Evidence that membrane‐bound melittin is monomeric
Authors: Altenbach Christian , Hubbell Wayne L.
Published: May 2005

Spin‐labeled derivatives of the bee venom protein, melittin, were obtained by reacting on the average one of the four amino groups of the protein with succinimidyl‐2,2,5,5‐tetramethyl‐3‐pyrroline‐1‐oxyl‐3‐carboxylate All 16 statistically possible reaction products with 0, 1, 2, 3 or 4 spin labels per protein were then separated in a single pass with reversed phase high performance liquid chromatography. With the help of trypsin digestion and diode array detection it was possible to assign the primary structure of all 16 eluting fractions. All fractions with only one spin label per protein were purified for electron paramagnetic resonance measurement. The labeling sites cover different regions of the protein: one is at the N‐terminus, one at lysine‐7, and two are near the C‐terminus at lysine‐21 and lysine‐23, respectively. This set of specifically labeled melittins was used to study the structure and dynamics of melittin in aqueous solutions and when bound to neutral or negatively charged membranes. In aqueous solution a reduction in rotational correlation time and appearance of spin‐spin interaction was observed during salt‐induced transition from a random coil monomer to a mostly α‐helical retramer. Membrane binding to phospholipid bilayers in low or high ionic strength was reflected only in a further decrease in mobility. The absence of any spin interaction in the membrane‐bound state suggests that melittin is monomeric under these conditions. All derivatives were able to detect these structural changes, but melittin labeled at the N‐terminal amino group was especially valuable. Because of postulated intramolecular hydrogen bonding, this label reflects directly the motion of the entire protein or tetramer. Broadening experiments with chromium oxalate show that all labeled sites are at least partially exposed to the aqueous phase when melittin is bound to membranes. This suggests that an α‐helical melittin monomer binds to membranes with its axis parallel to the membrane surface.
Preferred conformational state of the N‐terminus section of a bovine growth hormone fragment (residues 96‐133) in water is an omega loop
Authors: Gooley P. R. , Carter S. A. , Fagerness P. E. , MacKenzie N. E.
Published: May 2005

The solution structure of a 38‐amino‐acid‐residue, biologically active fragment of bovine growth hormone (bGH96–133) was investigated with a combined nuclear magnetic resonance (NMR) and computer modeling approach. With the distance geometry program DISGEO and distance constraints derived from the nuclear Overhauser enhancement (NOE) experiments, it was found that residues Ser‐100 to Tyr‐110 circumscribe an Ω‐loop, a recently categorized feature of nonregular secondary protein structure.
X‐ray and model‐building studies on the specificity of the active site of proteinase K
Authors: Betzel C. , Bellemann M. , Pal G. P. , Bajorath J. , Saenger W. , Wilson K. S.
Published: May 2005

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy‐Ala‐Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X‐ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least‐squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca2+ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three‐stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.
Structural analysis of the 2.8 Å model of xylose isomerase from Actinoplanes missouriensis
Authors: Rey Felix , Jenkins John , Janin Joël , Lasters Ignace , Alard Philippe , Classens Michel , Matthyssens Gaston , Wodak Shoshana
Published: May 2005

The structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 22 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight‐stranded α/β “T.I.M.” barrel forming an N‐terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The β barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair binding site for Eu3+ ions, which are assumed to the sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimmers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of the other α/β barrel enzymes.
Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli
Authors: Graddis Thomas J. , Klig Lisa S. , Yanofsky Charles , Oxender Dale L.
Published: May 2005

Availability of the three‐dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that from the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C‐tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of theronine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two‐and four‐fold, respectively. Replacement of ariginine 54 by histidine (RH84) or glycine 85 by ariginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants prduces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimer formation upon treatment at 65°C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L‐tryptophan. Indole‐3‐propionic acid, 5‐methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation. These results indicate that the presence of the indole moiety in the corepressor binding pocket increases the stability of the dimer.
Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes
Authors: Murphy Michael E. P. , Moult John , Bleackley R. Chris , Gershenfeld Howard , Weissman Irving L. , James Michael N. G.
Published: May 2005

Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for the cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three‐dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has been provided useful information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X‐ray structures of four serine proteinase to provide a structurally based sequences alignment: α‐chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat must cell protease 2 (RMCP2). The root mean square differences in α‐carbon atom positions among these four structures when compared in a pairwise fashions range form 0.79 to 0.97 Å for structurally equivalent residues. Te sequences of the two lymphocyte enzymes were then aligned to these proteinase using chemical criteria and the superimposed X‐ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. RmCP2 and BT as templates for HF, the molecular models were constructed. Intermolecular steric clashes that resulted from replacement of amino acid chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angels in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginie residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 positions of the substrate. Both CCP1 and HF have a free cysteine in the segment of the polypeptide 88 to 93. Models of the dimeric form of these enzymes can be constructed by forming disulfide bridges between the suitably oriented monomers, ie., Cys88–Cys88′ for CCP1 and Cys93–Cys93′ for HF.
Refined structure of human carbonic anhydrase II at 2.0 Å resolution
Authors: Eriksson A. Elisabeth , Jones T. Alwyn , Liljas Anders
Published: May 2005

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure–factor least‐squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc‐bound water, seven more water molecules are identified in the active site. Assuming that Glu‐106 is deprotonated at pH 8.5, some of the hydrogen bond donor–acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr‐199 donates its proton to the Oε1 atom of Glu‐106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.
Thermitase, a thermostable subtilisin: Comparison of predicted and experimental structures and the molecular cause of thermostability
Authors: Frömmel Cornelius , Sander Chris
Published: May 2005

The Subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, Subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three‐dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserve structural core and less well in seven loop regions involving insertions and deletions relative to Subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular case of thermostability we confront hypotheses erived by calculation of the details of interatomic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main cause of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.
Structural principles of α/β barrel proteins: The packing of the interior of the sheet
Authors: Lesk Arthur M. , Brändén Carl‐Ivar , Chothia Cyrus
Published: May 2005

α/β barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight‐stranded β‐sheets and the packing of the residues at the center of the barrel. The Packingin thisregion is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at someof the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues fromthe second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even‐numbered strands that form the central layer, and side chains from the oddnumbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed β barrel.
Rapid calculation of the solution scattering profile from a macromolecule of known structure
Authors: Lattman Eaton Edward
Published: May 2005

If one expands the structure factor equation in spherical coordinates, rotational averaging of the molecular Fourier transform, which leads directly to the solution scattering profile, is greatly simplified. It becomes a projection in the polar and azimuthal angular variables. The profile is given by

equation image

The index j runs over all atoms; r, θ, ϕ are atomic coordinates and ε and N are constants; the Ym,n are complex spherical harmonics, and Jn are spherical Bessel functions; R = 2 sin θ/λ. The effects of solvent have been modeled by subtracting from each protein atom a properly weighted water. Hydrogens have been included by using scattering curves fj derived from the spherical averaging ofprotein atoms with their attached hydrogens. This approach may also be satisfactory for neutron scattering. Published scattering profiles2 for lysozyme and BPTI have been accurately matched in less than one‐tenth the time required by other methods. Separate, adjustable temperature factors for the protein, solvent waters, and bound watersare used, and appear to be needed. In the case of BPTI, as suggested by NMR observations, the observed diffraction pattern was much better accounted for by including only 4 tightly bound waters rather than the roughly 60 seen by crystallography.
Secondary structural analysis of retrovirus integrase: Characterization by circular dichroism and empirical prediction methods
Authors: Lin Thy‐Hou , Quinn Thomas P. , Grandgenett Duane , Walsh Mary T.
Published: May 2005

The retrovirus integrase (IN) protein is essentialfor integration of viral DNA into host DNA. The secondary structure of thepurified IN protein from avian myeloblastosis virus was investigated by bothcircular dichroism (CD) spectroscopy and five empirical prediction methods. The secondary structures determined from the resolving of CD spectra through a least‐squares curve fitting procedure were compared with those predicted from four statistical methods, e.g., the Chou–Fasman, arnier–Osguthorpe–Robson, Nishikawa–Ooi, and a JOINT scheme which combined all three of these methods, plus a pure a priori one, the Ptitsyn–Finkelstein method. Among all of the methods used, the Nishikawa–Ooiprediction gave the closest match in the composition of secondary structureto the CD result, although the other methods each correctly predictedoneor more secondary structural group. Most of the α‐helix and β‐sheet states predicted by the Ptitsyn–Finkelstein methodwere in accord with the Nishikawa–Ooi method. Secondary structural predictions by the Nishikawa–Ooi method were extended further toinclude IN proteins from four phylogenetic distinct retroviruses. The structuralrelationships between the four most conserved amino acid blocks of these IN proteins were compared using sequence homology and secondary structure predictions.
The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+ and the inhibitor pdTp, refined at 1.65 Å
Authors: Loll Patrick J. , Lattman Eaton E.
Published: May 2005

The structure of a complex of staphylococcal nuclease with Ca2+ and deoxythymidine 3′,5′‐biophosphate (pdTp) has been refined by stereochemically restrained leastsquares minimization to a crystallographic R value of 0.161 at Å resolution. The estimated root‐mean‐square (rms) error in the coordinates in 0.16 Å. The final model comprises 1082 protein atoms, onecalcium ion, the pdTp molecule, and 82 protein atoms, onecalcium ion, the pdTp molecule, and 82 solvent water molecules;it displays an rms deviation from ideality of 0.017 Å for bond distances and 1.8° for bond angles.

The mean distance between corresponding α carbons in the refined and unrefined structures is 0.6 Å we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket.

The details of the calcium liganding and solvent structure in the activesite are clearly shown in the final electron density map. The structure ofthe catalytic site is consistent with mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry‐related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that enzyme and substrate.
PKB: A program system and data base for analysis of protein structure
Authors: Bryant Stephen H.
Published: May 2005

PKB is a computer program system that combines a data base of three‐dimensional protein structures with a series of algorithms for pattern recognition, data analysis, and graphics. By typing relatively simple commands the user may search the data base for instances of a structural motif and analyze in detail the set of individual structures that are found. The application of PKB to the study of protein folding is illustrated in three examples. The first analysis compares the conformations observed for a short sequential motif, sequences similar to the cell‐attachment signal Arg‐Gly‐Asp. The second compares sequences observed for a conformational motif, a 16‐residue βαβ unit. The third analysis considers a population of substructures containing ion‐pair interaction, examining the relationship offrequency of occurrence to calculated electrostatic energy.
The structure of aconitase
Authors: Robbins A. H. , Stout C. D.
Published: May 2005

The crystal structure of the 80,000 Da FeS enzyme aconitase has been solved and refined at 2.1 Å resolution. The protein contains four domains; the first three from the N‐terminus are closely associated around the [3Fe–4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Associationof the larger C‐terminal domain with the first three domains createsan extensive cleft leading to the FeS cluster. Residues from all four domains contribute to the active site region, which is defined by the FeS cluster and a bound SOion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well asseveral bound water molecules. Three of these side chains reside on a threeturn 310 helix in the first domain. The SOion is bound 9.3 Å from the center of the [3Fe–4S] cluster by the side chains of 2 Arg and 1 Gln rsidues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.
Evolution of CuZn superoxide dismutase and the Greek Key β‐barrel structural motif
Authors: Getzoff Elizabeth D. , Tainer John A. , Stempien Michelle M. , Bell Graeme I. , Hallewell Robert A.
Published: May 2005

Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron‐exon boundary locations, gene duplication, and Greek key β‐barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of uperoxide dismutation. The β‐strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the β‐strands and at both termini. Thus, the β‐barrel with only four invariant residues is apparently over determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility ofthe Greek key β‐barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.
Molecular dynamics simulations of ribonuclease T1: Comparison of the free enzyme and 2′ GMP–enzyme complex
Authors: MacKerell Alexander D , Nilsson Lennart , Rigler Rudolf , Heinemann Udo , Saenger Wolfram
Published: May 2005

Molecular dynamics simulations were performed on free RNase T1 and the 2′GMP–RNase T1 complex in vacuum and with water in the active site along with crystallographically identified waters, allowing analysis of both active site and overall structural and dynamics changes due to the presence of 2′GMP. Difference in the active site include a closing in the presence of 2′GMP, which is accompanied by a decrease in mobility of active site residues. The functional relevance of the active site fluctuations is discussed. 2′GMP alters the motion of Tyr‐45, suggesting a role for that residue in providing a hydrophobic environment for the protein–nucleic acid interactions responsible for the specificity of RNase T1. The presence of 2′GMP causes a structural change of the C‐terminus of the α‐helix, indicating the transmission of structural changes from the active site through the protein matrix. Overall fluctuations of both the free and 2′GMP enzyme forms are in good agreement with X‐ray temperature factors. The motion of Trp‐59 is influenced by 2′GMP, indicating difference in enzyme dynamics away from the active site, with the calculated changes following those previously seen in time‐resolved fluorescence experiments.
The effects of truncating long‐range forces on protein dynamics
Authors: Loncharich Richard J , Brooks Bernard R
Published: May 2005

This paper considers the effects of truncating long‐range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long‐range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom–atom truncation based on distance; (4) simple atom–atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group–group truncation based on distance; and (6) simple group–group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates of a no cutoff simulation and from the X‐ray structure of the protein. The simulations in which long‐range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 Å, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5–9 Å range gives reasonable results for a distance‐dependent dielectric, but not using a constant dielectric. Both the atom–atom and group–group truncation methods based on distance shows large rms deviations and fluctuation for short cutoff distance, while for cutoff distance of 11 Å or greater, reasonable results are achieved. Although comparison of these to distance‐based truncation methods show surprisingly larger rms deviations for the group–group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom–atom or group–group list‐based simulations generally appear to be less stable than the distance‐based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.
Salt or ion bridges in biological system: A study employing quantum and molecular mechanics
Authors: Deerfield David W. , Nicholas Hugh B. , Hiskey Richard G. , Pedersen Lee G.
Published: May 2005

Equilibrium geometries and binding energies of model “salt” or “ion” bridge systems have been computed by ab initio quantum chemistry techniques (GAUSSIAN82) and by empirical techniques (AMBER2.0). Formate and dimethyl phosphate served as anions in the model compounds while interacting with several organic cations, including methyl ammonium, methyl guanidinium, and divalent metal ion (either Mg2+ or Ca2+) without and with an additional chloride; and a divalent metal ion (either Mg2+ or Ca2+), chloride, and four water molecules of hydration about the metal ion. The majority of the quantum chemical computations were performed using a split‐valence basis set. For the model compounds studied we find that the ab initio geometries are in remarkably goodagreement with the molecular mechanics geometries.Several Calculations werealso performed using diffuse fractions. The formate anion binds these modelcations more strongly than does dimethyl phosphate, while the organiccation methyl ammonium binds model anions more strongly than does methyl guanidinium. Finally, in model compounds including organic anions, Mg2+ or Ca2+ and four molecules of water, and a chloride anion, we find that the equilibrium structure of the magnesium complex involves a solvent separated ion pair (the magnesium ion is six coordinate), whereas the calcium ion complex remains seven coordinate. Molecular mechanics overestimates binding energies, but the estimates may be close enough to actual binding energies togive useful insight into the details energies to give useful insight into the details of salt bridges in biological systems.
Peptide sequencing and site‐directed mutagenesis identify tyrosine‐319 as the active site tyrosine of Escherichia coli DNA topoisomerase I
Authors: Lynn Richard M. , Wang James C.
Published: May 2005

Tyrosine 319 of E. coli topoisomerase I is shown to be the activesite tyrosine that becomes covalently attached to a DNA 5′ phosphoryl group during the transient breakage of a DNA internucleotide bond by the enzyme. The tyrosine was mapped by trapping the covalent complex between the DNA and DNA topoisomerase I, digesting the complex exhaustively with trypsin, and sequencing the DNA‐linked tryptic peptide. Site‐directed mutagenesis converting Tyr‐319 to a serine or phenylalanine completely inactivates the enzyme. The structure of the enzyme andits catalysis of DNA strand breakage, passage, and rejoining are discussed in terms of the available information.
Engineering subtilisin BPN′ for site‐specific proteolysis
Authors: Carter Paul , Nilsson Björn , Burnier John P. , Burdick Daniel , Wells James A.
Published: May 2005

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN′, which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wildtype subtilisin BPN′ is greatly restricted by substitution of the catalytic histidine 64 with alanine (H64A) so that certain histidine‐containing substrates are preferentially hydrolysed (Carter, P., Wells, J. A. Science 237:394–399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20‐fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild‐type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N‐terminal sequence. Several features of H64A variants of subtilisin make them attractive for site‐specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N‐terminal side of the cleavage site and yet are broadly specific on the C‐terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.