摘要:

AbstractBy inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand‐binding sites on their membrane‐bound receptors. Three residues in a loop on the surface ofE. colialkaline phosphatase were substituted by an 18‐residue peptide containing the receptor‐binding segment of somatostatin‐14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand‐binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild‐type enzyme and the hormone‐enzyme hybrid displaced125I‐labeled somatostatin from rat brain membrane receptors only at high concentrations. How‐ever, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor‐binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild‐type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand‐binding site of somatostatin receptor

ISSN:0887-3585    

DOI:10.1002/prot.340140103

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe rate of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein–disulfide–isomerase. No effects of added ATP or Ca2+were observed, even though protein–disulfide–isomerase blinds Ca2+tightly. © 1992 Wiley

ISSN:0887-3585    

DOI:10.1002/prot.340140104

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

年代:1992

数据来源: WILEY

摘要:

AbstractSignature sequences are contiguous patterns of amino acis 10‐50 resiues long that are associated with a particular structure or function in proteins. These may be of three types (by our nomenclature): superfamily signatures, remnant homologies, and motifs. We have performed a systematic search through a database of protein sequences to automatically and preferentially find remnant homologies and motifs. This was accomplished in three steps:1We generated a nonredundant sequence database.2We used BLAST3 (Altschul and Lipman, Proc. Natl. Acad. Sci. U.S.A. 87:5509–‐5513, 1990) to generate local pairwise and triplet sequence alignments for every protein in the database vs. every other.3We selected “interesting” alignments and grouped them into clusters. We find that most of the clusters contain segments from proteins which share a common structure or function. Many of them correspond to signatures previously noted in the literature. We discuss three previously recognized motifs in detail (FAD/NAD‐binding, ATP/GTP‐binding, and cytochromeb5‐like domains) to demonstrate how the alignments generated by our procedure are consistent with previous work and make structural and functional sense. We also discuss two signatures (forN‐acetyltransferases and glycerol‐phosphate binding) which to our knowledge have not been previously recognized. ©

ISSN:0887-3585    

DOI:10.1002/prot.340140105

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

年代:1992

数据来源: WILEY

摘要:

AbstractA new analytical mutagenesis technique is described that involves randomining the DNA sequence of a short stretch of a gene (3–6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein. Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical. We applied this method to 66 codons of the gene encoding TEM‐1 β‐lactamase in 19 separate experiments. We found that TEM‐1 β‐lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild‐type enzyme. We also found a few exceptional regions where only a few random sequences function. Examination of the X‐ray structures of homologous β‐lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein. DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related β‐lactamases.

ISSN:0887-3585    

DOI:10.1002/prot.340140106

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

年代:1992

数据来源: WILEY

摘要:

AbstractMolecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate–enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermedite complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma. © 1992 Wiley‐Liss,

ISSN:0887-3585    

DOI:10.1002/prot.340140107

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe structure of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X‐ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the olecules, although the regions that display most variability are associated with the loop structures (residues 14–17 and 24–29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higherB‐factors, although it is not appearent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X‐ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H‐bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance, the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96‐psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X‐ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low‐energy conformers that have been preferentially selected by crystallization factors. © 19

ISSN:0887-3585    

DOI:10.1002/prot.340140108

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe X‐ray structure of the C30V/C51A disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed at 1.6 Å resolution. The mutant crystallizes in a cell having two molecules in the asymmetric unit. The packing environments of these two molecules are quite different, allowing for an assessment of which among the observed structural changes result from the mutation and which are produced by lattice packing considerations. The removal of the 30–51 disulfide bridge has little apparent affect on theB‐factors of segments of adjacent polypeptide chain, although there are distinct differences in the structure compared to wild‐type BPTI crystal structures. Both of the two C30V/C51A molecules show differences at the mutation site when compared to another 30–51 disulfide mutant, C30A/C51A, presumably due to the larger steric bulk of a valine versus an alanine at residue 30. A comparison of the two independent C30V/C51A molecules indicates that there are significant differences between them even at the site of mutation. The description of the specific structural differences of each molecule differs in detail and suggests different conclusions about the nature of structural perturbation near 30–51. In addition, when these two molecules are compared to two different wild‐type structures, which had been determined from different space groups, a somewhat different pattern of changes is observed. These findings indicate that crystal packing can influence the observed perturbations in mutant structures. © 1992

ISSN:0887-3585    

DOI:10.1002/prot.340140109

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe hypothesis that dihydrolipoamide dehydrogenases (E3s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E3s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E3s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E3s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E3s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E3. The cofactor FAD and the substrakes NAD and lipoic acid were also included in the model. Unexpectedly, the surface residues in the cleft that holds the lipoamide were found to be highly charged and predominantly acidic, allowing us to predict that the region around the lipoamide in the sub‐unit should be basic in nature. The molecular model can be tested by site‐directed mutagenesis of residues predicted to be in the dihydrolipoamide acetyltransferase subunit binding cleft. © 1992 Wiley‐Lis

ISSN:0887-3585    

DOI:10.1002/prot.340140110

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

年代:1992

数据来源: WILEY

摘要:

AbstractA new minimization procedure for the global optimization is cartesian coordinate space of the conformational energy of a polypeptide chain is presented. The Metropolis Monte Carlo minimization is thereby supplemented by a thermalization process, which is initiated whenever a structure becomes trapped in an area containing closely located local minima in the conformational space. The method has been applied to the endogenous opioid pentapeptide methionine enkephalin. Five among 13 different starting conformations led to the same apparent global minimum of an in‐house developed energy function, a type II′ reverse turn, the central residues of which are Gly‐3‐Phe‐4. A comparison between the ECEPP/2 global minimum conformation of methionine enkephalin and the apparent one achieved by the present method shows that minimum‐energy conformations having a certain similarity can be generated by relatively different force fields. © 1992 Wil

ISSN:0887-3585    

DOI:10.1002/prot.340140111

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

年代:1992

数据来源: WILEY

摘要:

AbstractContinuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behaviour of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor.The model differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the thrid, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation.For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X‐ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near‐equilibrium conformations yielded a new ensemble in which the conformation most similar to the X‐ray determined structure PTI4 had the lowest total free energy.Despite the simplicity of the continuum slvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of sovlent. © 1992 Wiley‐L

ISSN:0887-3585    

DOI:10.1002/prot.340140112

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

年代:1992

数据来源: WILEY