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  • 1985-1989  (3,097)
  • Biochemistry and Biotechnology  (3,097)
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 1-7 
    ISSN: 0887-3585
    Keywords: helix stabilization ; helix dipole ; charged group ; pH titration ; electrostatic interaction ; hydrogen bonding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Interactions between the α-helix peptide dipoles and charged groups close to the ends of the helix were found to be an important determinant of α-helix stability in a previous study.1 The charge on the N-terminal residue of the C-peptide from ribonuclease A was varied chiefly by changing the α-NH2 blocking group, and the correlation of helix stability with N-terminal charge was demonstrated. An alternative explanation for some of those results is that the succinyl and acetyl blocking groups stabilize the helix by hydrogen bonding to an unsatisfied main-chain NH group. The helix dipole model is tested here with peptides that contain either a free α-NH3+ α-COO- groups, and no other charged groups that would titrate with similar pKa's. This model predicts that α-NH3α-COO- groups are helix-destabilizingand that the destabilizing interactions are electrostatic in origin. The hydrogen bonding model predicts that α-NH3 and α-COO- groups are not themselves helix-destabilizing, but that an acetyl or amide blocking group at the N- or C- terminus, respectively, stabilizes the helix by hydrogen bonding to an unsatisfied main-chain NH or CO group.The results are as follows: (1) Removal of the charge from α-NH3 and α-COO- groups by pH titration stabilizes an α-helix. (2) The increase in helix stability on pH titration of these groups is close to the increase produced by adding an acetyl or amide blocking group. (3) The helix-stabilizing effect of removing the charge from α-NH3 and α-COO- groups by pH titration is screened by increasing the NaCl concentration, and therefore the effect is electrostatic in origin. (4) Replacing the C-terminal amide blocking group with a methylester blocking group, which cannot donate a hydrogen bond, causes little change in helix stability.
    Additional Material: 6 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 8-12 
    ISSN: 0887-3585
    Keywords: ribonuclease A ; protein deamidation ; protein conformation ; disulfide bonds ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determine. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds.Deamidation of the labile Asn-67 residue of RNase A was followedelectrophoretically and chromatographically. At 80°C, similar rates of deamidation were observed for the disulfidebonded form, which is thermally unfolded, and the reduced form. At 37°C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the β-turn of residues 66-68.
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  • 4
    ISSN: 0887-3585
    Keywords: template-assembled synthetic protein (TASP) ; 4-helix bundle ; β-barrel structure ; protein de novo design ; peptide synthesis ; peptide conformation ; orthogonal protection ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The construction of a template-assembled synthetic protein (TASP) designed to contain both a 4-helix bundle and a β-barrel as two folding “domains” is described. For the de novo design of proteins, amphiphilic helices (α) and β-sheets (β) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intarmolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8-(4α)(4β). The design of this new macromolecule was assisted by computer modeling, which suggested a low-energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid-phase synthesis of the “two-domain” TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.
    Additional Material: 6 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 22-37 
    ISSN: 0887-3585
    Keywords: sequence homology ; tertiary structure prediction ; molecular dynamics ; energy minimization ; hydrophobic interactions ; aromatic ring-ring interactions ; salt bridges ; calcium binding ; thermoactinomyces vulgaris ; extracellular protease ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 47-65 
    ISSN: 0887-3585
    Keywords: crystal growth ; helical conformation ; repetitive octapeptide ; icelike molecular surface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The active conformation of an icenucleation protein, whose major portion consists of a long polypeptide segment of nearly repetitive octapeptides, is predicted by the analyses of conformational energy and the mechanism of crystal growth. The protein ideally has an exact octapeptide repetition and is assumed to have a helical conformation. The present study searched for low-energy helical conformations and each of the obtained low-energy conformations examined as to whether it has a surface structure that can promote crystal formation. Two conformations obtained were good candidates for an ice nucleus. Both were found to have on their surfaces an arrangement of hydrogen-bonding sites, which fits well with those of hydrogen bonds in hexagonal ice crystal. Further, one of the two conformations had a hexagonal conformational symmetry consistent with the hexagonal ice crystal structure. The other conformation had apentagonal conformational symmetry that could enable the growth of an ice crystal-dendritic polycrystalline snow crystal-which grows on metastable cubic ice.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 38-46 
    ISSN: 0887-3585
    Keywords: reconstitution ; membrane protein folding/unfolding ; pH effects ; differential scanning calorimetry ; circular dichroism ; electron microscopy ; HPLC ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Thermal unfolding experiments on bacteriorhodopsin in mixed Phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 ± 13 Å in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation. We conclude that the tertiary structure of the bacteriorhodopsin monomer is essentially the same in micelles and the purple membrane. On the other hand, in the synthetic mixed micelle system, the packing between the nonnative amphiphiles and bacteriorhodopsin is probably not optimal, protein-protein interactions have been lost, and thehelical packing may be looser because the crystalline lattice is absent. Itis likely that a combination of these effects leads to the decreased stability of micellar bacteriorhodopsin.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 78-92 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 93-95 
    ISSN: 0887-3585
    Keywords: folding intermediate ; molten globule state ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Previous results from equilibrium and kinetic studies of the folding of bovine growth hormone (bGH) have demonstrated that bGH does not follow a simple two-step folding mechanism. These results are summarized and interpreted according to the “molten globule” model. The molten globule state of bGH is characterized as a folding intermediate which largely a-helical, retains a compact hydrodynamic radius, has packing of the aromatic side chains that is similar to the unfolded state, and possesses a solvent-exposed hydrophobic surface along helix 106127 that readily leads association.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 66-77 
    ISSN: 0887-3585
    Keywords: subunit interactions ; allosteric regulation ; solvent accessibility ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Although ionizable groups are known to play important roles in the assembly, catalytic, and regulatory mechanisms of Escherichia coli aspartate transcarbamylase, these groups have not been characterized in detail. We report the application of static accessibility modified Tanford-Kirkwood theory to model electrostatic effects associated with the assembly of pair of chains, subunits, and the holoenzyme. All of the interchain interfaces except R1-R6 are stabilized by electrostatic interactions by -2 to -4 kcal-m-1 at pH 8. The pH dependence of the electrostatic component of the free energy of stabilization of intrasubunit contacts (C1-C2 and R1-R6) is qualitatively different from that of intersubunit contacts (C1-C4, C1-R1, and C1-R4). This difference may allow the transmission of information across subunit interfaces to be selectively regulated. Groups whose calculated pK or charge changes as a result of protein-protein interactions have been identified and the results correlated with available information about their function. Both the 240s loop of the c chain and the region near the Zn(II) ion of the r chain contain clusters of ionizable groups whose calculated pK values change by relatively large amounts upon assembly. These pK changes in turn extend to regions of the protein remote from the interface. The possibility that networks of ionizable groups are involved in transmitting information between binding sites is suggested.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. i 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 97-103 
    ISSN: 0887-3585
    Keywords: protein conformation ; dynamics ; Monte Carlo simulation ; conformational energy ; minimization ; spin glass ; conformational substates ; conformational heterogeneity ; hierarchy in dynamics ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A computer experiment of protein dynamics is carried out, which consists of two steps: (1) A Monte Carlo simulation of thermal fluctuations in the native state of a globular protein, bovinepancreatic trypsin inhibitor; and (2) a simulation of the quick freezingof fluctuating conformations into energy minima by minimization of the energy of a number of conformations sampled in the Monte Carlo simulations sampled in the Monte Carlo simulation. From the analysis of results of the computer experiment is obtained the following picture of protein dynamics:multiple energy minima exist in the native state, and they are distributedin clusters in the conformational space. The dynamics has a hierarchical structure which has at least two levels. In the first level, dynamics is restricted within one of the clusters of minima. In the second, transitions occur among the clusters. Local parts of a protein molecule, side chains and local main chain segments, can take multiple locally stable conformations in the native state. Many minima result from combinations of these multiple local conformations. The hierarchical structure in the dynamics comes from interactions among the local parts. Protein moleculeshave two types of flexibility, each associated with elastic and plastic deformations, respectively.
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  • 14
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 104-112 
    ISSN: 0887-3585
    Keywords: conformational fluctuations ; conformational heterogeneity ; conformational energy ; hierarchical structure ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Conformational fluctuations in a globular protein, bovine pancreatic trypsin inhibitor, in the time range between picoseconds and nanoseconds are studied by a Monte Carlo simulation method. Multipleenergy minima are derived from sampled conformations by minimizing their energy. They are distributed in clusters in the conformational space. A hierarchical structure is observed in the simulated dynamics. In the time range between 10-14 and 10-10 seconds dynamics is well represented by a superposition of vibrational motions within an energy well with transitions among minima within each cluster. Transitions among clusters take place in the time range of nanoseconds or longer.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 113-124 
    ISSN: 0887-3585
    Keywords: conformational fluctuations ; Monte Carlo simulation ; conformational energy ; conformational heterogeneity ; side chain conformation ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An analysis is carried out of differences in the minimum energy conformations obtained in the previous paper by energy minimization starting from conformations sampled by a Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatictrypsin inhibitor. Main conformational differences in each pair of energy minima are found usually localized in several side chains and in a few localmain chain segments. Such side chains and local main chain segments are found to take a few distinct local conformations in the minimum energy conformations. Energy minimum conformations can thus be described in terms of combinations of these multiple local conformations.
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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 125-131 
    ISSN: 0887-3585
    Keywords: protein conformation ; dynamics ; Monte Carlo simulation ; conformational energy ; minimization ; plastic deformation ; conformational heterogeneity ; hierarchy in dynamics ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Differences in atom packing are studied in the minimum energy conformations derived from the record of the Monte Carlo simulation of conformational fluctuation in the native state of a globular protein, bovine pancreatic trypsin inhibitor. It is found that local deformations observed among the minima which are found in the previous paper are accompanied by rearrangement of atom packing. Spatial locations of the local deformations in the three-dimensional folded structure are also studied. It is foundthat the local deformations are distributed in space in several clusters inthe folded structure. The size and location of the clusters characterize the respective fluctuations of the first and the second levels observed in the simulation. In the fluctuations of the first level local deformations, each of which usually involves a few side chains and one main chain local segment, are thermally exited independently of each other near thesurface of the molecule. The observed fluctuation of the second level involves a cooperative deformation involving many side chains and local main chain segments all in one cluster, which goes though the core of the molecule. The collective local deformations observed both in the first and second levels are plastic in the sense that they are accompanied with rearrangement of atom packing.
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  • 17
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 139-148 
    ISSN: 0887-3585
    Keywords: protein architecture ; packing ; evolutionary relationships ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 18
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 132-138 
    ISSN: 0887-3585
    Keywords: protein conformation ; dynamics ; Monte Carlo simulation ; conformational energy ; minimization ; hierarchy in dynamics ; conformational heterogeneity ; flexibility ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Distances between centers of gravity of individual residues are compared among the minimum energy conformations derived from the recordof the Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatic trypsin inhibitor. It is found that local deformations originating from the multiplicity of localconformations cause deformations of the whole structure of the molecule in various ways, which can be classified into two types. Type 1:When a local deformation occurs in a region consisting of a few residues near the surfaceof the molecule, the whole shape of the molecule responds by deforming elastically. The magnitude of this deformation is in the range of thermalfluctuations calculated by the harmonic approximation around a singleminimum. Type 2: We have observed one case belonging to the second type in which local deformations occur cooperatively in an extended region. This regiongoes across the whole molecule and divide the remaining parts into two. Atom packing changes in and around the extended region of local deformations. For this reason deformation in this region is plastic. Relative locationand orientation between the divided two parts change very much. Deformationof the whole shape in this case, associated with the plastic deformationin an extended region, demonstrates that protein molecules have a flexibility beyond the harmonic limit.
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  • 19
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 149-155 
    ISSN: 0887-3585
    Keywords: solution scattering ; low-angle scattering ; spherical averaging ; spherical harmonics ; spherical Fourier transform ; bound water ; solvent structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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 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.
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  • 20
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 156-165 
    ISSN: 0887-3585
    Keywords: retrovirus integrase ; circular dichroism ; homologous proteins ; secondary structural predictions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 21
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 166-169 
    ISSN: 0887-3585
    Keywords: α-crystallin ; enthalpy ; entropy of solution ; light scattering ; second virial coefficient ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Light scattering studies were performed on bovine α-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of α-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of α-crystallin are positive. The Flory thetatemperature was found to be 271 K.
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  • 22
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 170-182 
    ISSN: 0887-3585
    Keywords: modeling ; flavodoxin ; structure prediction ; side chains ; database ; structure analysis ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The tertiary structure of flavodoxin has been model build from only the X-ray crystallographic α-carbon coordinates. Main-Chain atoms were generated from a dictionary of backbone structures. Side-chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge-based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 Å across all atoms to the native structure. Regular secondary structural elements were modeledmore accurately than other regions. About 40%of the ξ1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions.The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contact, and hydrogen-bonding patterns could identifypoorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.
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  • 23
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 24
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 202-210 
    ISSN: 0887-3585
    Keywords: enhanced stability ; λCro ; genetic suppression ; intracellular proteolysis ; antibody screen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A mutant Cro protein, which bears the Ile-30→ Leu substitution, is thermally unstable and degraded more rapidly than wildtype Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12°C to 14°C. These amino acid substitutions affect residues 16 and 26, which are substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild-type Cro.
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  • 25
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 183-201 
    ISSN: 0887-3585
    Keywords: crystallographic refinement ; restrained least-squares refinement ; Konnert-Hendrickson refinement ; phosphodiesterase ; protein structure ; enzyme mechanism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 26
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 211-217 
    ISSN: 0887-3585
    Keywords: tyrosin ; mutant protein ; amino acid substitution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: CD spectra in the aromatic region of a series of the mutant α-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25°C. The measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of thewild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CDvalues of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for oneband at 276.5 nm. these results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residuesat position 49.
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  • 27
    ISSN: 0887-3585
    Keywords: yeast hexokinase II ; dimerization ; in vivo functions ; glucose repression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form).This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short from protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similarto wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form ofthe enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminalamino acids of hexokinase II are not required in vivo either for phosporylation of hexoses or for glucose repression.
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  • 28
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 233-247 
    ISSN: 0887-3585
    Keywords: protein folding ; crystallographic data base ; structural analysis ; computer program system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 29
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 224-232 
    ISSN: 0887-3585
    Keywords: effective pore's radius ; α-ketoglutarate dehydrogenase complex ; branched chain α-keto acid dehydrogenase complex ; electron microscopy ; multienzyme complex ; two-dimensional ; electrophoresis ; multienzyme complex ; aggregation of Pyruvate dehydrogenase complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two-dimensional agarose gel electrophoresis (2d-AGE) (Serwer, P. et al. Anal. Biochem. 152: 339-345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range of PE value needed for multienzyme complexes (effective radius, R = 10-30 nm). Appropriate conditions wereestablished to measure R value ± 1% of the pyruvate (PDC), α-ketoglutarate (α-KGDC), and the branched chain α-keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the sizestandards. The PDC from bovine heart was found to have an R = 22.4 ± 0.2 nm following cross-linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d-AGE. All R values for the enzyme complexes studied were agreement with, though more accurate than, R valuesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had an R of 18.8 ± 0.2 nm using 2d-AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisibleto electron microscopy.
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  • 30
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    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 31
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 248-257 
    ISSN: 0887-3585
    Keywords: subunit interactions ; icosahedral capsid ; electrostatic potential ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The role of the electrostatic interactions in the stability of the icosahedral β60 capsid of heavy riboflavin synthase from Bacillus Subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interaction agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand-free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between β-subunits forming dimers, which connect the relatively stable pentamers in the β-60 capsid. The release of the ligand causesareduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due tothe titratable groups and α-helix macrodipoles has been calculated on the basic of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polardistribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of the positive potential “channels” that coincide with the channels constituted by the pentameric and trimeric β-subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme-substrate recognition.
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  • 32
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 259-265 
    ISSN: 0887-3585
    Keywords: cDNA expression ; deletion mutagenesis ; zymogen activation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cellsto secrete the enzymically inactive pro-protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro-peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro-peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.
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  • 33
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 266-270 
    ISSN: 0887-3585
    Keywords: crystallization ; purification ; crystals ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 × 0.3 × 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which in enzymatically active upon dissolution. These crystals belong to the monoclinic space group P21 and have unit cell parameters a =114.5 Å, b=139.6 Å, c=125.7 Å, β=98.1° Self-rotation function studies indicate that there are three molecules per asymmetricunit. The crystals diffract to at least 3.0 Å resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.
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  • 34
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 281-288 
    ISSN: 0887-3585
    Keywords: Hu protein ; integration host factor ; transcription factor 1 ; DNA bending protein ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The prokaryotic protein HU, integration host factor (IHF) from Escherichia coli and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high-resolution structure of HU, we propose a model for thisinteraction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.
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  • 35
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 271-280 
    ISSN: 0887-3585
    Keywords: antifluorescyl monoclonal antibody ; high-affinity binding site ; effects of MPD on hapten binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 Å resolution by molecularreplacement methods. The starting model was the refined 2.7 Å structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecules relative to other members of an idiotypic family of monoclonalantifluorescyl antibodies. The phenyl carboxyl group of fluorescein appearsto be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domainsjust below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.
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  • 36
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 289-312 
    ISSN: 0887-3585
    Keywords: aconitase ; iron-sulfur enzyme ; crystal structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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 SO42-ion. 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 SO42-ion 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.
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  • 37
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 313-321 
    ISSN: 0887-3585
    Keywords: protein ; electron transfer ; molecular dynamic simulations ; dielectric ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Electrostatic calculations have been carried out on a number of structural conformers of tuna cytochrome c Conformers were generated using molecular dynamics simulations with a range of solvent simulating, macroscopic dielectric formalisms, and one solvent model that explicitly included solvent water molecules. Structures generated using the lowest dielectric models were relatively tight, with-side chains collapsed on the surface, while those from the higher dielectric modelshad more internal and external fluidity, with surface side chains exploring a fuller range of conformational space. The average structure generated with the explicitly solvated model corresponded most closely with the crystal structure. Individual pK values, overall titration curves, and electrostatic potential surfaces were calculated for average structures and along each simulation. Differences between structural conformers within each simulation give rise to substantial changes in calculated local electrostatic interactions, resulting in pK value fluctuations for individual sites in the protein that very by 0.3-2.0 pK units from the calculated time average. These variations are due to the thermal side chain reorientations that produce fluctuations in charge site separations. Properties like overall titration curves and pH dependent stability are not as sensitive to side chain fluctuations within a simulation, but there are substantial effects between simulation due to markeddifferences in average side chain behavior. These findings underscore the importance of proper dielectric formalism in molecular dynamics simulations when used to generate alternate solution structures from a crystal structure, and suggest that conformers significantly removed from the averagestructure have altered electrostatic properties that may prove important inepisodic protein properties such as catalysis.
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  • 38
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 322-336 
    ISSN: 0887-3585
    Keywords: sequence conservation ; exon ; gene duplication ; protein folding ; structure-function ; X-ray structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 39
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    Proteins: Structure, Function, and Genetics 6 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 40
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 1-19 
    ISSN: 0887-3585
    Keywords: virus crystallography ; molecular dynamics ; hydrophobic pockets ; antiviral agents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: X-Ray diffraction data have been obtained for nine related antiviral agents (“WIN compounds”) while bound to human rhinovirus 14 (HRV14).These compounds can inhibit both viral attachment to host cells and uncoating. To calculate interpretable electron density maps it was necessary to account for (1) the low (∼60percnt;) occupancies of these compounds in the crystal, (2) the large (up to 7.9 Å) conformational changes induced at the attachment site, and (3) the incomplete diffraction data. Application of a density difference map technique, which exploits the 20-fold noncrystallographic redundancy in HRV14, resulted in clear images of the HRV14:WIN complexes. A real-space refinement procedure was used to fit atomic models to these maps.The binding site of WIN compounds in HRV14 is a hydrophobic pocket composed mainly from residues that form the β-barrel of VP1. Among rhinoviruses, the residues associated with the binding pocket are far more conserved than external residues and are mostly contained within regular secondary structural elements. Molecular dynamics simulations of three HRV14:WIN complexes suggest that portions of the WIN compounds and viral protein near the entrance of the binding pocket are more flexible than portions deeper within the β-barrel.
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  • 41
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 337-354 
    ISSN: 0887-3585
    Keywords: computer simulation ; fluctuations in proteins ; secondary structural dynamics ; lysozyme ; protein-substrate complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, β-sheet, and loop structural elements in free and substrate-bound hen egg-white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and β-sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of theloop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate-bound simulation. A loop region (residues 101-107) of the active site cleft had significantly reduced motion due to interactions withthe substrate. Moreover, part of a loop and a 310 helix (residues of 67-88) not in contact with the substrate showeda marked increase in fluctuations. That these differences in dynamics of free and substrate-bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuationsof the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain φ and ψ angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place eitherabruptly, in 1 psec or less, or with a diffusive character over 5-10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 Å rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface areaand the dynamics of the different structural elements.
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  • 42
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 355-373 
    ISSN: 0887-3585
    Keywords: protein ; structure ; prediction ; primary ; secondary ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of Cα coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set ofstandard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can “replace” about 76% ofall hexamers in well-refined known proteins with an error of less than 1 Å, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.
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  • 43
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 20-31 
    ISSN: 0887-3585
    Keywords: ribonuclease ; active site ; conformational change ; protein-nucleic ; acid interactions ; fluorescence depolarization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
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  • 44
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 46-60 
    ISSN: 0887-3585
    Keywords: macromolecular conformation ; protein folding ; helical axis ; secondary structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a general and mathematically rigorous algorithm which allows the helicoidal structure of a protein to be calculated starting from the atomic coordinated of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal parameters describing the location of each peptide unit. The parameters obtained form a complete and independent set and can therefore be used for analyzing, comparing, or reconstructing protein backbone geometry. This algorithm has been implemented in a computer program named P-Curve. Several examples of its possible applications are discussed.
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  • 45
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 61-69 
    ISSN: 0887-3585
    Keywords: peptides ; hormones ; amphipathic helix ; hydrophobic moment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Eisenberg's helical hydrophobic moment (〈μH〉) algorithm was applied to the analysis of the primary structure of amphipathic α-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue 〈μH〉 for the amphipathic helical and control peptides were 0.46 (±/-0.07) and 0.33 (0.07) (P+0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular 〈μH〉 was plotted vs 〈μH〉. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic α helix at residues 4-21. This computer-based method may enable rapid identification of peptide of the amphipathic α-helix class.
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  • 46
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 32-45 
    ISSN: 0887-3585
    Keywords: long range truncation ; molecular dynamics ; myoglobin ; truncation effects ; protein electrostatics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
    Additional Material: 7 Ill.
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  • 47
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 48
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 70-85 
    ISSN: 0887-3585
    Keywords: Protein electrostatics ; protein kinases ; effector protein ; calciumbinding protein ; α-helix ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain α-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-ldependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charges in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.
    Additional Material: 7 Ill.
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  • 49
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 87-103 
    ISSN: 0887-3585
    Keywords: molten globule state ; protein folding ; protein denaturation ; structural intermediate ; activated state of folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 50
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 104-127 
    ISSN: 0887-3585
    Keywords: lac repressor; lac operator ; lac headpiece-operator complex ; protein-DNA specificity ; molecular dynamics ; computer simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complexsatisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator based on many direct- and water-mediated hydrogenbonds and nonpolar contacts which allow the formation of a tight complex. Nostable hydrogen bonds between side chains and bases and found, while specific contacts occur between both nonpolar groups and, to a lesserextent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, andchemically induced dynamic nuclear polarization (CIDNP) data.
    Additional Material: 9 Ill.
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  • 51
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 128-138 
    ISSN: 0887-3585
    Keywords: DNA repair ; ultravioletlight ; pyrimidine dimers ; glycosylase ; apurinic/apyrimidinic endonuclease ; DNA repair enzymology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Previous structure/function analyses of the DNA repairenzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has beeninvestigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changesof Trp and Phe and more dramatic changes of Ser- a stop, codon, deletion of the codon or introduction of a frameshift. Both changesto the aromatic amino acids resulted in proteins which accumulated will in E. coli and not only significantly enhanced the UV survival of repair, deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129 → Trp and Tyr-129 → Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129 → Phe and Tyr-129 → Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129 → Phe mutant and to 20% that of wild type for the Tyr1-29 → Trp mutant.
    Additional Material: 4 Ill.
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  • 52
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 155-167 
    ISSN: 0887-3585
    Keywords: protein structure comparison ; dihedral angles ; protein conformation ; hemoglobin structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes Δt, the root mean square difference in φ and ψ torsion angles over a small number of amino acids (typically 3-5). Using this algorithm, large number of protein substrates comparisons were feasible. The parameter Δt was sensitive to variations in local protein conformation, and it correlates with Δr, the root mean square deviation in atomic coordinates. Values for Δt were obtained that define similarity thresholds, which determine whether two substructure are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of Δt due to measurement noise fromcomparisons of independently refined coordinates of the same protein. A sample distribution of Δt from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons large numbers of nonmatching comparisons. Unlike methods based on Cα atoms alone, Δt was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologus proteins by Δt showed that the active site torsion angles are highly conserved. The Δt method was applied to the α-chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.
    Additional Material: 8 Ill.
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  • 53
    ISSN: 0887-3585
    Keywords: retrovirus ; bacterial expression ; high-performance liquid chromatography ; NH2- and COOH-terminal sequence analysis ; kcat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala-13 to Gly-185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short-lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species isonly partially processed, and it, a 13 kDA intermediate, and the mature 11 kDA enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2- and COOH-terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2≅ 9 min) processes itself into a species with a mass of ∼13kDa and a species with a mass of ∼11 kDa. Both of these latter species can be separated by RP-HPLC, have the NH2-terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP-HPLC and SDS poly acrylamide gel electrophoresis. On extended incubation at 4°C at either neutral or acidic pH all species of the proteinexhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.
    Additional Material: 7 Ill.
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  • 54
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 55
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 215-215 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 56
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 168-192 
    ISSN: 0887-3585
    Keywords: salt bridge ; ab initio ; calcium ; magnesium ; carboxylate ; phosphate ; ammonium ; guanidinium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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.
    Additional Material: 16 Ill.
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  • 57
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 217-221 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: No abstract.
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  • 58
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 222-230 
    ISSN: 0887-3585
    Keywords: G proteins ; p21ras ; GTPase ; cholera toxin ; GTPase-activating protein ; amino acid sequence ; protein structure ; conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The functions of G proteins - like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras) - depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP- and GDP-bound states. Similarities in function and amino acid sequence indicate that EF-Tu, p21ras, and G protein α-chains evolved from a primordial GTP-binding protein. Proteins in all three families appear to share common mechanisms for GTP-dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the α-chain of Gs (αs) point to key regions that are involved in GTP-dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of αs in human pituitary tumors inhibit-the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in αs that corresponds to Gln-61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of the GTPase inhibiting mutations in αs occurs in the codon for an ARG residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by αs. This Arg residue is located in a domain of αs not represented in EF-Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF-Tu by the programmed ribosome and for p21ras by the recently discovered GTPase-activating protein. Owing to their inherited similarities of structure and function, what we learn about αs, p21ras, or EF-tu as individual molecules helps us to understand crucial functions of other members of the super-family.
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  • 59