bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of arginine 143 in the electrostatics and mechanism of Cu, Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis
Authors: Fisher Cindy L. , Cabelli Diane E. , Tainer John A. , Hallewell Robert A. , Getzoff Elizabeth D.
Published: May 2005

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active‐site Cu ion is rate‐limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence‐invariant Arg‐143 in human Cu, Zn superoxide dismutase, single‐site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg‐143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg‐143 by Lys caused a 2‐fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10‐fold, while charge‐reversing substitutions (Asp, Glu) caused a 100‐fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro‐tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild‐type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild‐type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild‐type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg‐143. Results from this joint analysis establish that, aside from the Cu ligands, Arg‐143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley‐Liss, Inc.
Crystallization of a fragment of human fibronectin: Introduction of methionine by site‐directed mutagenesis to allow phasing via selenomethionine
Authors: Leahy Daniel J. , Erickson Harold P. , Aukhil Ikramuddin , Joshi Paritosh , Hendrickson Wayne A.
Published: May 2005

Crystals of a fragment of human fibronectin encompassing the 7th through the RGD‐containing 10th type III repeats (FN7–10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 Å Bragg spacings. A mutant FN7–10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site‐directed mutagenesis. Diffraction‐quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site‐directed mutagenesis to allow phasing from selenomethionyl‐substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed. © 1994 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray crystallographic analysis of phospholipid transfer protein from maize seedlings
Authors: Shin Dong Hae , Hwang Kwang Yeon , Kim Kyeong Kyu , Kim Sangsoo , Sweet Robert M. , Suh Se Won
Published: May 2005

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P212121 with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.36 Å 3/Da and a solvent content of 48% by volume. The X‐ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuKα and synchrotron X‐rays. A set of X‐ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley‐Liss, Inc.
Recognition of distantly related proteins through energy calculations
Authors: Abagyan Ruben , Frishman Dmitrij , Argos Patrick
Published: May 2005

A new method to detect remote relationships between protein sequences and known three‐dimensional structures based on direct energy calculations and without reliance on statistics has been developed. The likelihood of a residue to occupy a given position on the structural template was represented by an estimate of the stabilization free energy made after explicit prediction of the substituted side chain conformation. The profile matrix derived from these energy values and modified by increasing the residue self‐exchange values successfully predicted compatibility of heatshock protein and globin sequences with the three‐dimensional structures of actin and phycocyanin, respectively, from a full protein sequence databank search. The high sensitivity of the method makes it a unique tool for predicting the three‐dimensional fold for the rapidly growing number of protein sequences. © 1994 Wiley‐Liss, Inc.
Accuracy of protein flexibility predictions
Authors: Vihinen Mauno , Torkkila Esa , Riikonen Pentti
Published: May 2005

Protein structural flexibility is important for catalysis, binding, and allostery. Flexibility has been predicted from amino acid sequence with a sliding window averaging technique and applied primarily to epitope search. New prediction parameters were derived from 92 refined protein structures in an unbiased selection of the Protein Data Bank by developing further the method of Karplus and Schulz (Naturwissenschaften 72:212–213, 1985). The accuracy of four flexibility prediction techniques was studied by comparing atomic temperature factors of known three‐dimensional protein structures to predictions by using correlation coefficients. The size of the prediction window was optimized for each method. Predictions made with our new parameters, using an optimized window size of 9 residues in the prediction window, were giving the best results. The difference from another previously used technique was small, whereas two other methods were much poorer. Applicability of the predictions was also tested by searching for known epitopes from amino acid sequences. The best techniques predicted correctly 20 of 31 continuous epitopes in seven proteins. Flexibility parameters have previously been used for calculating protein average flexibility indices which are inversely correlated to protein stability. Indices with the new parameters showed better correlation to protein stability than those used previously; furthermore they had relationship even when the old parameters failed. © 1994 Wiley‐Liss, Inc.
Molecular dynamics simulation of the docking of substrates to proteins
Authors: Di Nola Alfredo , Roccatano Danilo , Berendsen Herman J. C.
Published: May 2005

A simple method is described to perform docking of subtrates to proteins or probes to receptor molecules by a modification of molecular dynamics simulations. The method consists of a separation of the center‐of‐mass motion of the substrate from its internal and rotational motions, and a separate coupling to different thermal baths for both types of motion of the substrate and for the motion of the receptor. Thus the temperatures and the time constants of coupling to the baths can be arbitrarily varied for these three types of motion, allowing either a frozen or a flexible receptor and allowing control of search rate without disturbance of internal structure. In addition, an extra repulsive term between substrate and protein was applied to smooth the interaction. The method was applied to a model substrate docking onto a model surface, and to the docking of phosphocholine onto immunoglobulin McPC603, in both cases with a frozen receptor. Using transrational temperatures of the substrate in the range of 1300–1700 K and room temperature for the internal degrees of freedom of the substrate, an efficient nontrapping exploratory search (“helicopter view”) is obtained, which visits the correct binding sites. Low energy conformations can then be further investigated by separate search or by dynamic simulated annealing. In both cases the correct minima were identified. The possibility to work with flexible receptors is discussed. © 1994 Wiley‐Liss, Inc.
Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae
Authors: Villoutreix Bruno O. , Spassov Velin Z. , Atanasov Boris P. , Hervé Guy , Ladjimi Moncef M.
Published: May 2005

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three‐dimensional structure of this domain was generated on the basis of the known X‐ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X‐ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domain of the multifunctional protein. © 1994 Wiley‐Liss, Inc.
Torsion angle dynamics: Reduced variable conformational sampling enhances crystallographic structure refinement
Authors: Rice Luke M. , BrüNger Axel T.
Published: May 2005

A reduced variable conformational sampling strategy for macromolecules based on molecular dynamics in torsion angle space is evaluated using crystallographic refinement as a prototypical search problem. Bae and Haug's algorithm for constrained dynamics [Bae, D. S., Haug, E. J. A recursive formulation for constrained mechanical system dynamics. Mech. Struct. Mach. 15:359–382, 1987], originally developed for robotics, was used. Their formulation solves the equations of motion exactly for arbitrary holonomic constraints, and hence differs from commonly used approximation algorithms. It uses gradients calculated in Cartesian coordinates, and thus also differs from internal coordinate formulations. Molecular dynamics can be carried out at significantly higher temperatures due to the elimination of the high frequency bond and angle vibrations. The sampling strategy presented here combines high temperature torsion angle dynamics with repeated trajectories using different initial velocities. The best solutions can be identified by the free R value, or the R value if experimental phase information is appropriately included in the refinement. Applications to crystallographic refinement show a significantly increased radius of convergence over conventional techniques. For a test system with diffraction data to 2 Å resolution, slow‐cooling protocols fail to converge if the backbone atom root mean square (rms) coordinate deviation from the crystal structure is greater than 1.25 Å, but torsion angle refinement can correct backbone atom rms coordinate deviations up to approximately 1.7 Å. © 1994 Wiley‐Liss, Inc.
Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences
Authors: Magnus Karen A. , Hazes Bart , Ton‐That Hoa , Bonaventura Celia , Bonaventura Joseph , Hol Wim G. J.
Published: May 2005

The X‐ray structure of an oxygenated hemocyanin molecule, subunit II of Limulus polyphemus hemocyanin, was determined at 2.4 Å resolution and refined to a crystallographic R‐factor of 17.1%. The 73‐kDa subunit crystallizes with the symmetry of the space group R32 with one subunit per asymmetric unit forming hexamers with 32 point group symmetry. Molecular oxygen is bound to a dinuclear copper center in the protein's second domain, symmetrically between and equidistant from the two copper atoms. The copper‐copper distance in oxygenated Limulus hemocyanin is 3.6 ± 0.2 Å, which is surprisingly 1 Å less than that seen previously in deoxygenated Limulus polyphemus subunit II hemocyanin (Hazes et al., Protein Sci. 2:597, 1993). Away from the oxygen binding sites, the tertiary and quaternary structures of oxygenated and deoxygenated Limulus subunit II hemocyanins are quite similar. A major difference in tertiary structures is seen, however, when the Limulus structures are compared with deoxygenated Panulirus interruptus hemocyanin (Volbeda, A., Hol, W. G. J. J. Mol. Biol. 209:249, 1989) where the position of domain 1 is rotated by 8° with respect to domains 2 and 3. We postulate this rotation plays an important role in cooperativity and regulation of oxygen affinity in all arthropod hemocyanins. © 1994 Wiley‐Liss, Inc.
A prediction of the secondary structure of the pleckstrin homology domain
Authors: Jenny Thomas F. , Benner Steven A.
Published: May 2005

A consensus prediction for the secondary structure of the pleckstrin homology (PH) domain is presented. The prediction is based on an analysis of patterns of conservation and variation of homologous protein sequences. The structure is predicted to be formed largely from beta strands with a single alpha helix. © 1994 Wiley‐Liss, Inc.
Protein stability parameters measured by hydrogen exchange
Authors: Bai Yawen , Milne John S. , Mayne Leland , Englander S. Walter
Published: May 2005

The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equilibrium. These rates can be measured by one‐dimensional nuclear magnetic resonance and used to determine the thermodynamic parameters of global unfolding at mild solution conditions well below the melting transition. The free energy for global unfolding measured by hydrogen exchange can differ from values found by standard denaturation methods, most notably due to the slow cis‐trans isomerization of the prolyl peptide bond. This difference can be quantitatively calculated from basic principles. Even with these corrections, HX experiments at low denaturant concentration measure a free energy of protein stability that rises above the usual linear extrapolation from denaturation data, as predicted by the denaturant binding model of Tanford. © 1994 Wiley‐Liss, Inc.
A special‐purpose computer for molecular dynamics: GRAPE‐2A
Authors: Ito Tomoyoshi , Fukushige Toshiyuki , Makino Junichiro , Ebisuzaki Toshikazu , Okumura Sachiko K. , Sugimoto Daiichiro , Miyagawa Hiroo , Kitamura Kunihiro
Published: May 2005

Molecular dynamics simulations have been extensively used in research of proteins. Since these simulations are quite computer intensive, their acceleration is of main interest of the research. In molecular dynamics simulations, almost all computing time is consumed in calculating the forces between particles, e.g., Coulomb and van der Waals forces. We have designed and built GRAPE‐2A (GRAvity PipE 2A), a special‐purpose computer for use in simulations of classical many‐body systems. GRAPE‐2A calculates forces exerted on a particle from the other particles. GRAPE‐2A can calculate force of an arbitrary functional form of a central force. The host computer, which is connected to GRAPE‐2A through the VME bus, performs other calculations such as time integration. The peak speed of GRAPE‐2A is 180 Mflops. We can also stimulate systems with periodic boundary conditions by the Ewald method, using GRAPE‐2A and another special‐purpose computer, WINE (Wave space INtegrator for the Ewald method). © 1994 Wiley‐Liss, Inc.
Coiled‐coil stutter and link segments in keratin and other intermediate filament molecules: A computer modeling study
Authors: North Anthony C. T. , Steinert Peter M. , Parry David A. D.
Published: May 2005

Structural discontinuities have previously been identified in four regions of the coiled‐coil rod domain structure present in intermediate filament (IF) protein molecules. These include a point at which a phase shift occurs in the heptad periodicity characteristic of the sequence of polar and apolar residues in α‐helical coiled‐coils, and three links that lack a heptad substructure. We have studied these regions by computer‐based molecular modeling and comparative sequence analysis and conclude that the phasing discontinuity can be accommodated without significant distortion of the overall double‐helical chain conformation; the L2 link has a similar conformation in all different types of IF molecules, a favorable conformation being one in which the two strands wrap tightly around each other; the L12 links vary in length between different IF types but contain important sequence similarities suggestive of a partial β structure; the L1 links show larger variations in length, a lower degree of similarity, and probably diverse structures. Variations in the overall charges of the different links suggest that ionic interactions may playa significant role in filament assembly. The results also have general significance for other α‐fibrous proteins in which either the characteristic heptad phasing undergoes a discontinuity or where a short non‐coiled‐coil sequence occurs within a coiled‐coil rod domain structure. © 1994 Wiley‐Liss, Inc.
Intrinsic secondary structure propensities of the amino acids, using statistical ϕ–ψ matrices: Comparison with experimental scales
Authors: Muñoz Victor , Serrano Luis
Published: May 2005

Today there are several different experimental scales for the intrinsic α‐helix as well as β‐strand, propensities of the 20 amino acids obtained from the thermodynamic analysis of various model systems. These scales do not compare well with those extracted from statistical analysis of three‐dimensional structure databases. Possible explanations for this could be the limited size of the databases used, the definitions of intrinsic propensities, or the theoretical approach. Here we report a statistical determination of α‐helix and β‐strand propensities derived from the analysis of a database of 279 three‐dimensional structures. Contrary to what has been generally done, we have considered a particular residue as in α‐helix or β‐strand conformation by looking only at its dihedral angles (ϕ–ψ matrices). Neither the identity nor the conformation of the surrounding residues in the amino acid sequence has been taken into consideration. Pseudoenergy empirical scales have been calculated from the statistical propensities. These scales agree very well with the experimental ones in relative and absolute terms. Moreover, its correlation with the average of the experimental scales for α‐helix or β‐strand is as good as the correlations of the individual experimental scales with the average. These results show that by using a large enough database and a proper definition for the secondary structure propensities, it is possible to obtain a scale as good as any of experimental origin. Interestingly the ϕ–ψ analysis of the Ramachandran plot suggests that the amino acids could have different β‐strand propensities in different subregions of the β‐strand area. © 1994 Wiley‐Liss, Inc.
Hydrophobic docking: A proposed enhancement to molecular recognition techniques
Authors: Vakser Ilya A. , Aflalo Claude
Published: May 2005

In the classical procedures for predicting the structure of protein complexes two molecules are brought in contact at multiple relative positions, the extent of complementarity (geometric and/or energy) at the surface of contact is assessed at each position, and the best fits are retrieved. In view of the higher occurrence of hydrophobic groups at contact sites, their contribution results in more intermolecular atom–atom contacts per unit area for correct matches than for false positive fits. The hydrophobic groups are also potentially less flexible at the surface. Thus, from a practical point of view, a partial representation of the molecules based on hydrophobic groups should improve the quality of the results in finding molecular recognition sites, as compared to full representation. We tested this proposal by applying the idea to an existing geometric fit procedure and compared the results obtained with full vs. hydrophobic representations of molecules in known molecular complexes. The hydrophobic docking yielded distinctly higher signal‐to‐noise ratio so that the correct match is discriminated better from false positive fits. It appears that nonhydrophobic groups contribute more to false matches. The results are discussed in terms of their relevance to molecular recognition techniques as compared to energy calculations. © 1994 Wiley‐Liss, Inc.
A P‐loop‐like motif in a widespread ATP pyrophosphatase domain: Implications for the evolution of sequence motifs and enzyme activity
Authors: Bork Peer , Koonin Eugene V.
Published: May 2005

A conserved amino acid sequence motif was identified in four distinct groups of enzymes that catalyze the hydrolysis of the α–β phosphate bond of ATP, namely GMP synthetases, argininosuccinate synthetases, asparagine synthetases, and ATP sulfurylases. The motif is also present in Rhodobacter capsulata AdgA, Escherichia coli NtrL, and Bacillus subtilis OutB, for which no enzymatic activities are currently known. The observed pattern of amino acid residue conservation and predicted secondary structures suggest that this motif may be a modified version of the P‐loop of nucleotide binding domains, and that it is likely to be involved in phosphate binding. We call it PP‐motif, since it appears to be a part of a previously uncharacterized ATP pyrophophatase domain. ATP sulfurylases, NtrL, and OutB consist of this domain alone. In other proteins, the pyrophosphatase domain is associated with amidotransferase domains (type I or type II), a putative citrulline‐aspartate ligase domain or a nitrilase/amidase domain. Unexpectedly, statistically significant overall sequence similarity was found between ATP sulfurylase and 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) reductase, another protein of the sulfate activation pathway. The PP‐motif is strongly modified in PAPS reductases, but they share with ATP sulfurylases another conserved motif which might be involved in sulfate binding. We propose that PAPS reductases may have evolved from ATP sulfurylases; the evolution of the new enzymatic function appears to be accompanied by a switch of the strongest functional constraint from the PP‐motif to the putative sulfate‐binding motif. © 1994 Wiley‐Liss, Inc.
Thermodynamic genetics of the folding of the B1 immunoglobulin‐binding domain from streptococcal protein G
Authors: O'Neil Karyn T. , Hoess Ronald H. , Raleigh Daniel P. , DeGrado William F.
Published: May 2005

A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG‐binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG‐binding function, it is possible to identify mutants that are folded but destabilized compared with wild‐type Strp G. Such mutants can be divided into three general categories: (1) those that disrupt packing of hydrophobic side chains in the protein interior; (2) those that destabilize secondary structure; and (3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild‐type Strp G but stabilities that were decreased by 2–5 kcal/mol. © 1995 Wiley‐Liss, Inc.
A vector projection method for predicting the specificity of GalNAc‐transferase
Authors: Chou Kuo‐Chen , Zhang Chun‐Ting , Kézdy Ferenc J. , Poorman Roger A.
Published: May 2005

The specificity of UDP‐Gal‐NAc:polypeptide N‐acetylgalactosaminytransferase (GalNAc‐transferase) is consistent with the existence of an extended site composed of nine subsites, denoted by P4, P3, P2, P1, P0, P1′, P2′, P3′, and P4′, where the acceptor at P0 is being either Ser or Thr. To predict whether a peptide will react with the enzyme to form a Ser‐ or Thr‐conjugated glycopeptide, a vector projection method is proposed which uses a training set of amino acid sequences surrounding 90 Ser and 106 Thr O‐glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. The model postulates independent interactions of the 9 amino acid moieties with their respective binding sites. The high ratio of correct predictions vs. total predictions for the data in both the training and the testing sets indicates that the method is self‐consistent and efficient. It provides a rapid means for predicting O‐glycosylation and designing effective inhibitors of GalNAc‐transferase. © 1995 Wiley‐Liss, Inc.
Structural studies of a synthetic peptide derived from the carboxyl‐terminal domain of RNA polymerase II
Authors: Cagas Perseveranda M. , Corden Jeffry L.
Published: May 2005

The conformation of the repeating heptapeptide unit of the carboxyl‐terminal domain of RNA Polymerase II, Y1S2P3T4S5P6S7 has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56‐residue peptide H2N‐(S2P3T4S5P6S7Yl)8‐COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β‐turns located at Ser2‐Pro3‐Thr4‐Ser5 (SPTS) and Ser5‐Pro6‐Ser7‐Tyr1 (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry‐simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56‐mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56‐mer. Twenty‐seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β‐turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser2, Ser5, Thr4, and Ser7 all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl‐terminal domain with a regular but unusual secondary structure consisting of a series of staggered β‐turns. © 1995 Wiley‐Liss, Inc.
Computational approaches to study protein unfolding: Hen egg white lysozyme as a case study
Authors: Hünenberger P. H. , Mark A. E. , van Gunsteren W. F.
Published: May 2005

Four methods are compared to drive the unfolding of a protein: (1) high temperature (T‐run), (2) high pressure (P‐run), (3) by imposing a gradual increase in the mean radius of the protein using a penalty function added to the physical interaction function (F‐run, radial force driven unfolding), and (4) by weak coupling of the difference between the temperature of the radially outward moving atoms and the radially inward moving atoms to an external temperature bath (K‐run, kinetic energy driven unfolding). The characteristic features of the four unfolding pathways are analyzed in order to detect distortions due to the size or the type of the applied perturbation, as well as the features that are common to all of them. Hen egg white lysozyme is used as a test system. The simulations are analyzed and compared to experimental data like 1H‐NMR amide proton exchange‐folding competition, heat capacity, and compressibility measurements. © 1995 Wiley‐Liss, Inc.
A model for the LexA repressor DNA complex
Authors: Knegtel Ronald M. A. , Fogh Rasmus H. , Boelens Rolf , Kaptein Robert , Ottleben Gunnar , Rüterjans Heinz , Dumoulin Pascal , Schnarr Manfred
Published: May 2005

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein–DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B‐DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn‐41, Glu‐44, and Glu‐45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser‐39, Ala‐42, and Asn‐41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two β‐sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half‐site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed. © 1995 Wiley‐Liss, Inc.
The first solvation shell of magnesium ion in a model protein environment with formate, water, and X‐NH3, H2S, imidazole, formaldehyde, and chloride as ligands: An ab initio study
Authors: Deerfield David W. , Fox Douglas J. , Head‐Gordon Martin , Hiskey Richard G. , Pedersen Lee G.
Published: May 2005

The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH3, H2S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate–Mg(II) ion is greater than 10 kcal mol−1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH3 formaldehyde, H2O, and H2S. © 1995 Wiley‐Liss, Inc.
LINUS: A hierarchic procedure to predict the fold of a protein
Authors: Srinivasan Rajgopal , Rose George D.
Published: May 2005

We describe LINUS, a hierarchic procedure to predict the fold of a protein from its amino acid sequence alone. The algorithm, which has been implemented in a computer program, was applied to large, overlapping fragments from a diverse test set of 7 X‐ray‐elucidated proteins, with encouraging results. For all proteins but one, the overall fragment topology is well predicted, including both secondary and supersecondary structure. The algorithm was also applied to a molecule of unknown conformation, groES, inwhich X‐ray structure determination is presently ongoing. LINUS is an acronym for Local Independently Nucleated Units of Structure. The procedure ascends the folding hierarchy in discrete stages, with concomitant accretion of structure at each step. The chain is represented by simplified geometry and folds under the influence of a primitive energy function. The only accurately described energetic quantity in this work is hard sphere repulsion–the principal forceinvolved in organizing protein conformation [Richards, F. M. Ann. Rev. Biophys. Bioeng. 6:151–176, 1977]. Among other applications, the method is a natural tool for use in the human genome initiative. © 1995 Wiley‐Liss, Inc.
Structural model of the photosynthetic reaction center of Rhodobacter capsulatus
Authors: Foloppe Nicolas , Ferrand Michel , Breton Jacques , Smith Jeremy C.
Published: May 2005

The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis. However, no three‐dimensional structure is available for this protein. We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations. In the Rb. Capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps. Viridis and Rb. Sphaeroides. The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements. A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps. viridis, Rb. sphaeroides, and Rb. capsulatus. © 1995 Wiley‐Liss, Inc.
Cloning and expression of the uracil–DNA glycosylase inhibitor (UGI) from bacteriophage PBS‐1 and crystallization of a uracil–DNA glycosylase–UGI complex
Authors: Savva Renos , Pearl Laurence H.
Published: May 2005

The uracil‐DNA glycosylase inhibitory protein (UGI) from the bacterio‐phage PBS‐l has been cloned and overexpressed. The nucleotide sequence is identical to that for the previously described PBS‐2 inhibitor. The recombinant PBS‐l UGI inhibits the uracil‐DNA glycosylase from herpes simplex virus type‐l (HSV‐l UDGase), and a complex between the HSV‐l UDGase and PBS‐l UGI has been crystallized. The crystals have unit cell dimensions a = 143.21 Å, c = 40.78 Å and are in a polar hexagonal space group. There is a single complex in the asymmetric unit with a solvent content of 62% by volume and the crystals diffract to 2.5Å on a synchrotron radiation source. © 1995 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray analysis of the cytoplasmic domain of human erythrocyte band 3
Authors: Kiyatkin Anatoly B. , Natarajan Padmaja , Munshi Sanjeev , Minor Wladyslaw , Johnson John E. , Low Philip S.
Published: May 2005

A cytoplasmic domain of the human erythrocyte membrane protein band 3 (Mr = 42,500), residues 1–379, expressed in and purified from E. coli, has been crystallized by the method of vapor diffusion in sitting drops with subsequent streak‐seeding at room temperature. Initial crystals were grown from solutions containing 65–68% saturated ammonium sulfate at pH 4.9 and 2 mg/ml protein. Subsequent streak‐seeding into solutions of 50–53% ammonium sulfate at pH 4.9 and 7 mg/ml protein produced single crystals suitable fur X‐ray analysis, which contained pure protein as revealed by gel electrophoresis. The crystals belong to the monoclinic space group C2 with cell dimensions of a = 178.8 Å, b = 90.5 Å, c = 122.1 Å, and β = 131.3° and diffract at least to 2.7 Å resolution (at 100 K). A self‐rotation function shows the presence of approximate 222 local symmetry. © 1995 Wiley‐Liss, Inc.
A predicted consensus structure for the protein kinase C2 homology (C2H) domain, the repeating unit of synaptotagmin
Authors: Gerloff Dietlind L. , Chelvanayagam Gareth , Benner Steven A.
Published: May 2005

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2‐sheet beta sandwich structure, with one of only three of the many possible folds being preferred. © 1995 Wiley‐Liss, Inc.
Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei
Authors: Reinikainen Tapani , Teleman Olle , Teeri Tuula T.
Published: May 2005

Cellobiohydrolase I (CBHI) is the major cellulase of Trichoderma reesei. The enzyme contains a discrete cellulose‐binding domain (CBD), which increases its binding and activity on crystalline cellulose. We studied cellulase‐cellulose interactions using site‐directed mutagenesis on the basis of the three‐dimensional structure of the CBD of CBHI. Three mutant proteins which have earlier been produced in Saccharomyces cerevisiae were expressed in the native host organism. The data presented here support the hypothesis that a conserved tyrosine (Y492) located on the flat and more hydrophilic surface of the CBD is essential for the functionality. The data also suggest that the more hydrophobic surface is not directly involved in the CBD function. The pH dependence of the adsorption revealed that electrostatic repulsion between the bound proteins may also control the adsorption. The binding of CBHI to cellulose was significantly affected by high ionic strength suggesting that the interaction with cellulose includes a hydrophobic effect. High ionic strength increased the activity of the isolated core and of mutant proteins on crystalline cellulose, indicating that once productively bound, the enzymes are capable of solubilizing cellulose even with a mutagenized or with no CBD. © 1995 Wiley‐Liss, Inc.
Activation of blood coagulation factor VIIa with cleaved tissue factor extracellular domain and crystallization of the active complex
Authors: Kirchhofer Daniel , Guha Arabinda , Nemerson Yale , Konigsberg William H. , Vilbois Francis , Chène Christiane , Banner David W. , D'Arcy Allan
Published: May 2005

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active‐site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS‐PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.‐C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N‐ and C‐terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N‐ and C‐terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 × 0.12 × 0.6 mm and showed diffraction to a resolution of 3 Å. © 1995 Wiley‐Liss, Inc.
Circular dichroism of the parallel β helical proteins pectate lyase C and E
Authors: Sieber Volker , Jurnak Frances , Moe Gregory R.
Published: May 2005

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel β‐helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel β‐sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel β‐sheets these proteins provide a unique opportunity to study the effect of parallel β‐helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca2+, derive the parallel β‐helical components of the spectra, and compare these results with previous CD studies of parallel β‐sheet structure. The shape and intensity of the parallel β‐sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel β‐helical folding motif. © 1995 Wiley‐Liss, Inc.
Local moves: An efficient algorithm for simulation of protein folding
Authors: Elofsson Arne , Le Grand Scott M. , Eisenberg David
Published: May 2005

We have enhanced genetic algorithms and Monte Carlo methods for simulation of protein folding by introducing “local moves” in dihedral space. A local move consists of changes in backbone dihedral angles in a sequential window while the positions of all atoms outside the window remain unchanged. We find three advantages of local moves: (1) For some energy functions, protein conformations of lower energy are found; (2) these low energy conformations are found in fewer steps; and (3) the simulations are less sensitive to the details of the annealing protocol. To distinguish the effectiveness of local move algorithm from the complexity of the energy function, we have used several different energy functions. These energy functions include the Profile score (Bowie et al., Science 253:164–170, 1991), the knowledge‐based energy function used by Bowie and Eisenberg 1994 (Proc. Natl. Acad. Sci. U.S.A. 91:4434–4440, 1994), two energy terms developed as suggested by Sippl and coworkers (Hendlich et al., J. Mol. Biol. 216:167180, 1990), and AMBER (Weiner and Kollman, J. Comp. Chem. 2:287‐303, 1981). Besides these energy functions we have used three energy functions that include knowledge of the native structures: the RMSD from the native structure, the distance matrix error, and an energy term based on the distance between different residue types called DBIN. In some of these simulations the main advantage of local moves is the reduced dependence on the details of the annealing schedule. In other simulations, local moves are superior to other algorithms as structures with lower energy are found. © 1995 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction studies of complexes between an influenza hemagglutinin and fab fragments of two different monoclonal antibodies
Authors: Gigant Benoît , Fleury Damien , Bizebard Thierry , Skehel John J. , Knossow Marcel
Published: May 2005

Fab fragments from two different monoclonal antibodies (BH151 and HC45) which bind to the same antigenic region of the influenza hemagglutinin were crystallized as complexes with the hemagglutinin. The complexes crystallize in PEG 600, pH 6.0, and PEG 2000, pH 8.5, respectively. Both crystals belong to space group P321, with very similar unit cell dimensions. © 1995 Wiley‐Liss, Inc.
Why do protein architectures have boltzmann‐like statistics?
Authors: Finkelstein Alexei V. , Badretdinov Azat Ya. , Gutin Alexander M.
Published: May 2005

A theoretical study has shown that the occurrence of various structural elements in stable folds of random copolymers is exponentially dependent on the own energy of the element. A similar occurrence‐on‐energy dependence is observed in globular proteins1 from the level of amino acid conformations to the level of overall architectures. Thus, the structural features stabilized by many random sequences are typical of globular proteins while the features rarely observed in proteins are those which are stabilized by only a minor part of the random sequences. © 1995 Wiley‐Liss, Inc.
Harmonicity and anharmonicity in protein dynamics: A normal mode analysis and principal component analysis
Authors: Hayward Steven , Kitao Akio , Gō Nobuhiro
Published: May 2005

A comparison of a normal mode analysis and principal component analysis of a 200‐ps molecular dynamics trajectory of bovine pancreatic trypsin inhibitor in vacuum has been made in order to further elucidate the harmonic and anharmonic aspects in the dynamics of proteins. An anharmonicity factor is defined which measures the degree of anharmonicity in the modes, be they principal modes or normal modes, and it is shown that the principal mode system naturally divides into anharmonic modes with peak frequencies below 80 cm−1, and harmonic modes with frequencies above this value. In general the larger the mean‐square fluctuation of a principal mode, the greater the degree of anharmonicity in its motion. The anharmonic modes represent only 12% of the total number of variables, but account for 98% of the total mean‐square fluctuation. The transitional nature of the anharmonic motion is demonstrated. The results strongly suggest that in a large subspace, the free energy surface, as probed by the simulation, is approximated by a multi‐dimensional parabola which is just a resealed version of the parabola corresponding to the harmonic approximation to the conformational energy surface at a single minimum. After 200 ps, the resealing factor, termed the “normal mode resealing factor,” has apparently converged to a value whereby the mean‐square fluctuation within the subspace is about twice that predicted by the normal mode analysis. © 1995 Wiley‐Liss, Inc.
Three‐dimensional model of the α‐subunit of bacterial luciferase
Authors: Sandalova Tatyana , Lindqvist Ylva
Published: May 2005

The predicted secondary structure of both subunits of bacterial luciferase is in accordance with a regular 8‐fold α/β‐barrel structure. The 3D profile1,2 confirmed that luciferase subunits are compatible with the α/β‐barrel despite the absence of sequence similarity with any α/β‐barrel protein. The three‐dimensional structure of 260 residues of the α‐chain of luciferase was modeled from coordinates of glycolate oxidase and then energy minimized. The model obtained satisfies the criteria for the structure of a globular protein and is in accordance with known experimental data. From the model it is possible to predict active site residues involved in binding and catalysis. These predictions, and thus also the model, can be tested by protein engineering experiments. © 1995 Wiley‐Liss, Inc.