摘要:

AbstractThe conformation of the staphylococcal nuclease‐bound metal–dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425–7437] was docked into the X‐ray structure of the enzyme–Ca2+‐3′,5′‐pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183–201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr‐85, Tyr‐113, and Tyr‐115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme–La3+‐dTdA complex were sequence specifically assigned by 2D phase‐sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronu‐clear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY‐HMQC spectros‐copy with15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme–La3+–3′,5′‐pdTp complex and the enzyme–Ca2+–3′,5′‐pdTp complex, proton resonances of Tyr‐85, Tyr‐113, Tyr‐115, and Phe‐34 were shifted by 0.08 to 0.33 ppm and the15N resonance of Tyr‐113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme‐bound dTdA shows the 5′‐dA leaving group to partially overlap the inhibitor, 3′,5′‐pdTp (in the X‐ray structure). Tne 3′‐TMP moiety of dTdA points toward the solvent in a channel defined by Ile‐18, Asp‐19, Thr‐22, Lys‐45, and His‐46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu‐43 and to attack the phosphorus with inversion. Arg‐35 and Arg‐87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg‐87 positioned to protonate the leaving 5′‐oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5′‐dGMP onto the 3′‐oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu‐80, Asp‐83, Lys‐84, and Tyr‐115 with its 3′‐OH group accessibl

ISSN:0887-3585    

DOI:10.1002/prot.340130402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractMolecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such asd‐phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 Å centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 Å sphere of water molecules. MD calculations were carried out for 45–60 ps. There is no large deviation from the available X‐ray structures of native and the dPhe adduct for the MD stuctures. Average MD structures were calculated starting from the X‐ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed. © 1992 Wiley

ISSN:0887-3585    

DOI:10.1002/prot.340130403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractStructures of the protein, transforming growth factor α (TGF‐α), have been derived from NMR data using distance geometry and subsequent energy refinement. Analysis of the sequential NOE distance bounds using a template algorithm provides a check for consistency in the calculation of bounds, stereospecific assignment of prochiral centers, and secondary structure assignment. Application of the template algorithm to the long range NOEs found within the NMR data sets collected at pH 6.3 and pH 3.4 is used to assess the confidence levels for the accuracy of the structures obtained from modeling. The method also provides critical insight in differentiating regions of the structure that are well defined from those that are not. Use of the restraint analysis protocol is shown to be a powerful adjunct to currently used methods for the assignment of protein structures from NMR data. © 1992 Wiley‐Lis

ISSN:0887-3585    

DOI:10.1002/prot.340130404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe accessible surface areas of 53 high‐resolution globin helices are correlated with molecular weight. The linear fit is assessed for satistical accuracy using a boot‐strap analysis, and by comparison to the areas of 13 ideal polyalanine α‐helices. The accessible area of the unfolded helices is compared with the folded values before helix‐helix packing. An analytical physical model is presented to explain the correlation, and to provide an analytical value for the surface area parameter in the diffusion‐collision model of protein folding. © 1992 Wile

ISSN:0887-3585    

DOI:10.1002/prot.340130405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe three‐dimensional structure of one of the three lipoamide dehydroge‐nases occurring inPseudomonas putida, LipDH Val, has been determined at 2.45 Å resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2‐fold axis generates the dimer which is observed in solution. The final crystallographicR‐factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+atoms, while the overallB‐factor is unusually high; 47 Å2.The structure of LipDH Val reveals the conformation of the C‐terminal residues which fold “back” into the putative lipoamide binding region. The C‐terminus has been proven to be important for activity by site‐directed mutagene‐sis. However, the distance of the C‐terminus to the catalytically essential residues is surprisingly large, over 6 Å, and the precise role of the C‐terminus still needs to be elucidated.In this crystal form LipDH Val contains one NAD+molecule per subunit. Its adenine‐ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide‐ribose moiety is far removed from its expected position near the isoallox‐azine ring and points into solution.Comparison of LipDH Val withAzotobacter vinelandiilipoamide dehydrogenase yields an rms difference of 1.6 Å for 440 well defined Cαatoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 Å with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed inAzotobacter vinelandiiLipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all

ISSN:0887-3585    

DOI:10.1002/prot.340130406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractAmino acid substitution analysis within a highly conserved region ofEscherichia colithymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure‐function relationship of theEscherichia coliTS protein at the N‐terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β‐strand. We find that residues surrounding a β‐bulge structure within the β‐strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wi

ISSN:0887-3585    

DOI:10.1002/prot.340130407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractPancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X‐ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombicI222 orI212121with unit cell parametersa= 54.38 Å,b= 72.29 Å, andc= 180.85 Å. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 Å.The far‐UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% α‐helix, between 10 and 20% antiparallel β‐strand, 10% parallel β‐strand, 15% turn, and 25 to 40% of other structures. © 199

ISSN:0887-3585    

DOI:10.1002/prot.340130408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340130401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY