bluuebunny/crossref_metadata_2025_split huggingface dataset:
Site specific point mutation changes specificity: A molecular modeling study by free energy simulations and enzyme kinetics of the thermodynamics in ribonuclease T1 substrate interactions
Authors: Elofsson Arne , Kulinski Tadeusz , Rigler Rudolf , Nilsson Lennart 
Published: May 2005

AbstractWe have theoretically and experimentally studied the binding of two different ligands to wild‐type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu‐46 replaced by Gln. The binding of the natural substrate 3′‐GMP has been compared with the binding of a fluorescent probe, 2‐aminopurine 3′‐monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild‐type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1‐Gln‐46 binds 2AP better than 3′GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i.e., the perturbation from Glu to Gln, than in the perturbation from 3′‐GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength. © 1993 Wiley‐Liss, Inc.
Binding of cyanide, cyanate, and thiocyanate to human carbonic anhydrase II
Authors: Peng Z. , Merz Kenneth M. , Banci Lucia 
Published: May 2005

AbstractComputer simulation techniques are used to address the question of how cyanide and related ions interact with human carbonic anhydrase II (HCAII). Spectroscopic results have suggested that cyanide is coordinated with the zinc ion, while recent X‐ray results suggest that the cyanide ion is noncovalently associated with the zinc–water or zinchydroxide form of the enzyme. We have carried out simulations on three models in an attempt to shed light on why the spectroscopic and X‐ray results differ. The first model we studied (Model I) has cyanide directly coordinated to the zinc ion, the second has it noncovalently interacting with the zinc–hydroxide (high pH) form of the enzyme (Model II), and the third has cyanide noncovalently interacting with the zinc–water (low pH) form of the enzyme (Model III). None of these models is satisfactory in explaining the available structural data obtained from X‐ray crystallography. This leads us to propose an alternative model, in which HCAII hydrates HCN to form an OH−/HCN complex coordinated to the Zn ion. Ab initio calculations are consistent with this model. Based on these results we are able to explain the observed crystallographic behavior of cyanate and, by inference, thiocyanate. © 1993 Wiley‐Liss, Inc.
Crystal structure of the complex of human α‐thrombin and nonhydrolyzable bifunctional inhibitors, hirutonin—2 and hirutonin—6
Authors: Zdanov Alexander , Wu Shan , DiMaio John , Konishi Yasuo , Li Yunge , Wu Xinjie , Edwards Brian F. P. , Martin Philip D. , Cygler Miroslaw 
Published: May 2005

AbstractThe crystal structure of the complexes of hirutonin‐2 and hirutonin‐6 with human α‐thrombin have been solved and refined to R‐factors of 0.169 (2.0 Å resolution) and 0.162 (201Å), respectively. Hirutonins belong to a family of bifunctional inhibitors bearing a noncleavable moiety mimicking the scissile bond. Hirutonin‐2 is an analog of (D)Phe‐Pro‐Arg‐Gly‐hirudin49–65; hirutonin‐6 has the same N‐terminal tripeptide connected to a shortened fibrinogen exosite‐binding part by a short, non‐peptidyl linker. The hirutonin‐6 molecule is well defined in the electron density with the exception of the C‐terminal Leu‐h61. The linker follows near the bottom of the canyon connecting the active site with the exosite, forms a short antiparallel β‐sheet‐like arrangement with Leu‐40‐Leu41 and makes van der Waals contacts with Glu39‐Leu40‐Leu41 of thrombin. In the thrombin‐hirutonin‐2 complex, the N‐ and C‐terminal parts of the inhibitor are well or dered (except the C‐terminal Gln‐h65) while the central portion of the linker is partially disordered. The glycine analog in the P1′ position of hirutonin‐2 assumes a conformation similar to that of the canonical form (Bode and Huber (1992) Eur. J. Biochem. 204 : 433‐451) and supports the identification of the S1′ site as restricted by His57, Trp60D, Lys60F, and the Cys42‐Cys58 disulfide bridge. The carbonyl oxygen of the P1 arginine residue is located in the oxyanion hole formed by the NH groups of Gly193 and Ser195, while the carbonyl carbon is positioned within a short distance, 2.8 Å, from the Oγ of Ser195. This resembles the conformation of the substrate‐like inhibitors bound to other serine proteases. The N‐terminal (D)Phe‐pro‐Arg fragment common to both inhibitors binds to thrombin in a fashion very similar to that of other inhibitors having this motif. The binding of the C‐terminus of hirutonins to the fibrinogen‐binding exosite is similar to that observed in hirudin and hirulog complexes. © 1993 Wiley‐Liss, Inc.
Single‐site modifications of half‐ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer
Authors: LiCata Vince J. , Dalessio Paula M. , Ackers Gary K. 
Published: May 2005

AbstractThe patterns of energetic response elicited by single‐site hemoglobin mutations and chemical mocdifications have been determined in order to probe the dimer–dimer interface of the half‐ligated tetramer (species[21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules1. In this study the free energies of quaternary assembly(dimers to tetramers) were determined for aseries of 24 tetrameric species in which one dimeric half‐molecule is ligated while the adjacent αβ dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half‐ligated tetramers and the unligated tetramers over the entire spatial distrubution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer–dimer interface of the half‐ligated tetramer(species[21]) has the same quaternary sturcture as that of the unligated molecule, i.e, “quaternary T.” This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T → R quaternary switchpoints2. Analysis of the observed Symmetry Rule behaviour in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between α and β subunits of the dimeric half‐tetramer within quaternary T. The mutational results indicate that these interactions are not “paid for” by breaking or making noncovalent bonds at the dimer–dimer interface (α1β2). They arise from structural and energetic changes that are “internal” to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of “tertiary constraint” is thus generated by the first binding step and is propagated to the second hemesite while the dimer–dimer interface α1β2serves as a constraint. The “sequential” cooperativity that occurs within the half‐molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers. When the constraint is released functionally by T to R dimer rearrangement (at each switch‐point specified by the a Symmetry Rule) the alterations of interfacial bonds then dominate the energetics of cooperativity. © 1993 Wiley‐Liss, Inc.
A calculation strategy for the structure determination of symmetric demers by 1H NMR
Authors: Nilges Michael 
Published: May 2005

AbstractThe structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter‐ and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving ther ambuguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamic‐based simulated annealing approach to minimize a traget function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the structure of a homologous protein is known and in ab intio structure determination. The method can be extended to higher order symmetric multimers. © 1993 Wiley‐Liss, Inc.
Preparation and characterization of the E168Q site‐directed mutant of yeast enolase 1
Authors: Brewer John M. , Robson Robert L. , Glover Claiborne V. C. , Holland Michael J. , Lebioda Lukasz 
Published: May 2005

AbstractYeast has two enolase isozymes (called 1 and 2), either of which suffices for growth. We cloned DNA encoding the enolase 1 protein coding and promoter regions flanked by BamHI termini using the PCR. The DNA, which contained no nucleotide base changes altering the protein sequence, was cloned into the multicopy shuttle vector pRS314 and transformed into a yeast strain with a deletion in its enolase 1 gene. The resulting plasmid‐containing strain makes enolase 1 in quantities which depend on cell growth. A ‘charge shuttle’ mechanism of action of enolase based on X‐ray crystallographic evidence (Lebioda and Stec, Biochemistry 30 : 2817, 1991) involves Glu‐168 accepting a proton from a water molecule that in turn accepts a proton from a carbon‐2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide‐directed site‐directed mutagenesis. Its identity was confirmed by N‐terminal sequence analysis, HPLC on Superose 12, SDS‐gel electrophoresis, and the sequence of the mutated DNA protein‐coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds substrate/product, AEP (3‐aminoenolpyruvate‐2‐phosphate, the 3‐amino analogue of the product phosphoenolpyruvate) and TSP (D‐tartronate semialdehyde‐2‐phosphate, the aldehyde analogue of the substrate 2‐phosphoglycerate), the latter two at least with affinities similar to those of the native enzyme. The E168Q enolase also produces absorbance changes in the analogues. The reaction with AEP is consistent with the ‘charge shuttle’ mechanism; the reaction with TSP, which presumably requires proton removal from carbon‐2, is complex but shows a very slow phase consistent with expectations. © 1993 Wiley‐Liss, Inc.
Entropy in biological binding processes: Estimation of translational entropy loss
Authors: Murphy Kenneth P. , Xie Dong , Thompson Kelly S. , Amzel L. Mario , Freire Ernesto 
Published: May 2005

AbstractThe loss of translational degrees of freedom makes an important, unfavorable contribution to the free energy of binding. Examination of experimental values suggest that calculation of this entropy using the Sackur–Tetrode equation produces largely overestimated values. Better agreement is obtained using the cratic entropy. Theoretical considerations suggest that the volumes available for the movement of a ligand in solution and in a complex are rather similar, suggesting also that the cratic entropy provides the best estimate of the loss of translational entropy. © 1994 John Wiley & Sons, Inc.
A Connected‐cluster of hydration around myoglobin: Correlation between molecular dynamics simulations and experiment
Authors: Lounnas Valère , Pettitt B. Montgomery 
Published: May 2005

AbstractAn analysis of a molecular dynamics simulation of metmyoglobin in an explicit solvent environment of 3,128 water molecules has been performed. Both statics and dynamics of the protein‐solvent interface are addressed in a comparison with experiment. Three‐dimensional density distributions, temperature factors, and occupancy weights are computed for the solvent by using the trajectory coordinates. Analysis of the hydration leads to the localization of more than 500 hydration sites distributed into multiple layers of solvation located between 2.6 and 6.8 Å from the atomic protein surface. After locating the local solvent density maxima or hydration sites we conclude that water molecules of hydration positions and hydration sites are distinct concepts. Both global and detailed properties of the hydration cluster around myoglobin are compared with recent neutron and X‐ray data on myoglobin. Questions arising from differences between X‐ray and neutron data concerning the locations of the protein‐bound water are investigated. Analysis of water site differences found from X‐ray and neutron experiments compared with our simulation shows that the simulation gives a way to unify the hydration picture given by the two experiments. © 1994 John Wiley & Sons, Inc.
Distribution function implied dynamics versus residence times and correlations: Solvation shells of myoglobin
Authors: Lounnas Valère , Pettitt B. Montgomery 
Published: May 2005

AbstractThe dynamics of water at the protein–solvent interface is investigated through the analysis of a molecular dynamics simulation of metmyoglobin in explicit aqueous environment. Distribution implied dynamics, harmonic and quasiharmonic, are compared with the simulated macroscopic dynamics. The distinction between distinguishable solvent molecules and hydration sites developed in the previous paper is used. The simulated hydration region within 7 Å from the protein surface is analyzed using a set of 551 hydration sites characterized by occupancy weights and temperature B‐factors determined from the simulation trajectory. The precision of the isotropic harmonic and anisotropic harmonic models for the description of proximal solvent fluctuations is examined. Residence times and dipole reorientation times of water around the protein surface are compared with NMR and ESR results. A correlation between diffraction experiment quantities such as the occupancy weights and temperature factors and the residence and correlation times resulting from magnetic resonance experiments is found via comparison with simulation. © 1994 John Wiley & Sons, Inc.
Crystallization and preliminary x‐ray diffraction studies of a monoclonal antibody fab fragment against foot‐and‐mouth disease virus and of its complex with the main antigenic site peptide
Authors: Verdaguer Nuria , Mateu Mauricio G. , Bravo Jerónimo , Tormo José , Giralt Ernest , Andreu David , Domingo Esteban , Fita Ignacio 
Published: May 2005

AbstractThe Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot‐and‐mouth disease virus (FMDV) C‐SBcl and its complex with a peptide, corresponding to the major antigenic site of FMDV (VPl residues 136–150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 Å resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 Å, b = 89.12 Å, c = 64.04 Å, and β = 112.9° and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 Å resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 Å, b = 60.67 Å, c = 143.45 Å, and β = 95.4°, Density packing considerations indicate that there are two Fab molecules in the asymmetric unit. © 1994 John Wiley & Sons, Inc.
Treatment of electrostatic effects in proteins: Multigrid‐based newton iterative method for solution of the full nonlinear poisson–boltzmann equation
Authors: Holst Michael , Kozack Richard E. , Saied Faisal , Subramaniam Shankar 
Published: May 2005

AbstractThe nonlinear Poisson–Boltzmann equation (NPBE) provides a continuum description of the electrostatic field in an ionic medium around a macromolecule. Here, a novel approach to the solution of the full NPBE is developed. This robust and efficient algorithm combines multilevel techniques with a damped inexact Newton's method. The CPU time required for solution of the full NPBE, which is less than that for standard single‐grid approaches in solving the corresponding linearized equation, is proportional to the number of unknowns enabling applications to very large macromolecular systems. Convergence of the method is demonstrated for a variety of protein systems. Comparison of the solutions to the linearized Poisson–Boltzmann equation shows that the damping of the electrostatic field around the charge is increased and that the potential scales logarithmically with charge. The inclusion of the full nonlinearity thus reduces the impact of highly charged residues on protein surfaces and provides a more realistic representation of electrostatic effects. This is demonstrated through calculation of potential around the active site regions of the 1,266‐residue tryptophan synthase dimer and in the computation of rate constants from Brownian dynamics calculations in the superoxide dismutase–superoxide and antibody–antigen systems. © 1994 John Wiley & Sons, Inc.
Cyanomet human hemoglobin crystallized under physiological conditions exhibits the Y quaternary structure
Authors: Smith Francine R. , Simmons Katharine C. 
Published: May 2005

AbstractCyanomet human hemoglobin has been crystallized at a chloride ion concentration and pH similar to physiological conditions. Molecular replacement calculations definitively show that the hemoglobin subunits are arranged in the Y quaternary form recently discovered in carbon monoxy hemoglobin Ypsilanti (99 Asp–Tyr), and subsequently observed in carbon monoxy normal human hemoglobin crystallized at low ionic strength and low pH. The structure has been refined at 2.09 Å resolution to an R‐value of 0.232, and further refinement is currently underway. Although the refinement is not yet complete, our results are the first indication that the Y structure may represent an important quaternary form of liganded hemoglobin under physiological buffer conditions. These results suggest the need for a reexamination of structure–function correlations in the hemoglobin system. © 1994 John Wiley & Sons, Inc.
α‐Helix‐forming propensities in peptides and proteins
Authors: Creamer Trevor P. , Rose George D. 
Published: May 2005

AbstractMuch effort has been invested in seeking to understand the thermodynamic basis of helix stability in both peptides and proteins. Recently, several groups have measured the helix‐forming propensities of individual residues (Lyu, P. C., Liff, M. I., Marky, L. A., Kallenbach, N. R. Science 250:669–673, 1990; O'Neil, K. T., DeGrado, W. F. Science 250:646–651, 1990; Padmanabhan, S., Marqusee, S., Ridgeway, T., Laue, T. M., Baldwin, R. L. Nature (London) 344:268–270, 1990). Using Monte Carlo computer simulations, we tested the hypothesis that these differences in measured helix‐forming propensity are due primarily to loss of side chain conformational entropy upon helix formation (Creamer, T. P., Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 89:5937–5941, 1992). Our previous study employed a rigid helix backbone, which is here generalized to a completely flexible helix model in order to ensure that earlier results were not a methodological artifact. Using this flexible model, side chain rotamer distributions and entropy losses are calculated and shown to agree with those obtained earlier. We note that the side chain conformational entropy calculated for Trp in our previous study was in error; a corrected value is presented. Extending earlier work, calculated entropy losses are found to correlate strongly with recent helix propensity scales derived from substitutions made within protein helices (Horovitz, A., Matthews, J. M., Fersht, A. R. J. Mol. Biol. 227:560–568, 1992; Blaber, M., Zhang, X.‐J., Matthews, B. M. Science 260:1637–1640, 1993). In contrast, little correlation is found between these helix propensity scales and the accessible surface area buried upon formation of a model polyalanyl α‐helix. Taken in sum, our results indicate that loss of side chain entropy is a major determinant of the helix‐forming tendency of residues in both peptide and protein helices. © 1994 Wiley‐Liss, Inc.
Crystallization and characterization of colicin E1 channel‐forming polypeptides
Authors: Elkins Patricia A. , Song Ho Yeong , Cramer William A. , Stauffacher Cynthia V. 
Published: May 2005

AbstractCrystals of the channel‐forming domain of colicin E1 from E. coli were grown by vapor diffusion at pH 6.4 and higher pH values. Cleavage of the colicin molecule with trypsin or thermolysin produced two of the pore‐forming polypeptides used in these experiments. The third polypeptide was purified from a constructed plasmid that overexpresses only the C‐terminal domain of colicin E1. Polypeptide crystals are tetragonal with space group I4, have one monomer in the asymmetric unit, and diffract to 2.2–2.4 Å. Unit cell parameters for the tryptic and thermolytic polypeptides are a = 102.9 Å and c = 35.6 Å. Crystals of the overexpressed polypeptide have unit cell parameters of a =87.2 Å and c =59.1 Å. The crystals were characterized by precession photography, and native data sets of each channel‐forming fragment were collected on a Siemens‐Nicolet area detector. The crystallization and characterization of these polypeptides are the first steps in the structure determination of the channel‐forming domain of colicin E1. © 1994 Wiley‐Liss, Inc.
The closed conformation of a highly flexible protein: The structure of E. coli adenylate kinase with bound AMP and AMPPNP
Authors: Berry Michael B. , Meador Bill , Bilderback Tim , Liang Peng , Glaser Michael , Phillips George N. 
Published: May 2005

AbstractThe structure of E. coli adenylate kinase with bound AMP and AMPPNP at 2.0 Å resolution is presented. The protein crystallizes in space group C2 with two molecules in the asymmetric unit, and has been refined to an R factor of 20.1% and an Rfree of 31.6%. In the present structure, the protein is in the closed (globular) form with the large flexible lid domain covering the AMPPNP molecule. Within the protein, AMP and AMPPNP, an ATP analog, occupy the AMP and ATP sites respectively, which had been suggested by the most recent crystal structure of E. coli adenylate kinase with AP5A bound (Müller and Schulz, 1992, ref. 1) and prior fluorescence studies (Liang et al., 1991, ref. 2). The binding of substrates and the positions of the active site residues are compared between the present structure and the E. coli adenylate kinase/Ap5A structure. We failed to detect a peak in the density map corresponding to the Mg2+ ion which is required for catalysis, and its absence has been attributed to the use of ammonium sulfate in the crystallization solution. Finally, a comparison is made between the present structure and the structure of the heavy chain of muscle myosin. © 1994 Wiley‐Liss, Inc.