摘要:

AbstractSingle alanine and glycine insertions were introduced at 20 randomly selected positions in staphylococcal nuclease. The resulting changes in catalytic activity and in stability to guanidine hydrochloride denaturation indicate that the native state structure is frequently able to accommodate the extra residue without great difficulty, even insertions within secondary structural elements such as alpha helices and beta sheets. On average, an inserted residue reduces the free energy of denaturation (ΔG H 2O) by an amount roughly comparable to an alanine or glycine substitution for one of the residues flanking the site of insertion. Several positions outside of the enzyme active site were found where insertions, but not substitutions, lead to structural changes that modify catalytic activity and the circular dichroism spectrum. Amino acid insertions represent a virtually unexplored class of genetic mutation that may prove complementary to amino acid substitutions for engineering proteins with altered functional and structural proper

ISSN:0887-3585    

DOI:10.1002/prot.340070402

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

年代:1990

数据来源: WILEY

摘要:

AbstractA method of targeted random mutagenesis has been used in investigate the informational content of 25 residue positions in two α‐helical regions of the N‐terminal domain of λ repressor. Examination of the functionally allowed sequences indicates that there is a wide range in tolerance to amino acid substitution at these position. At position that are buried in the structure, there are severe limitations on the number and type of residues allowed. At most surface positions, many different residues and residue types are tolerated. However, at several surface positions there is a string preference for hydrophilic amino acids, and at one surface position proline is absolutely conserved. The results reveal the high level of degeneracy in the information that specifies a particular protein

ISSN:0887-3585    

DOI:10.1002/prot.340070403

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

年代:1990

数据来源: WILEY

摘要:

AbstractComparative modeling methods are described that can be used to construct a three‐dimensional model structure of a new protein from knowledge of its sequence and of the experimental structure and sequences of other members of its homology family. The methods are illustrated with the mammalian serine protease family, for which seven experimental structures have been reported in the literature, and the sequence for over 35 different protein members of the family are available. The strategy for modeling these proteins is presented, and criteria are developed for determining and assigning the reliability of the modeled structure. Criteria are described that are specially designed to help detect cases in which it is likely that the local structure diverges significantly from the usual conformation of the famil

ISSN:0887-3585    

DOI:10.1002/prot.340070404

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

年代:1990

数据来源: WILEY

摘要:

AbstractVariant of the serine protease, subtilisin BPN′, in which the catalytic triad residues (Ser‐221, His‐64, and Asp‐32) are replaced singly or in combination by alanine retain activities with the substrateN‐succinyl‐L‐Ala‐L‐Ala‐L‐Pro‐L‐Phe‐p‐nitroanilide (sAAPF‐pna) that are at at least 103to 104above the non‐enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564–568, 1988]. A possible source of the residual activity was the hydrogen bond with the Nδ2of Asn‐155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild‐type enzyme. Replacing Asn‐155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF‐pnaby 150‐fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcatis actually 5‐foldgreaterthan for the S221A enzyme. Thus, the catalytic role of Asn‐155 is dependent upon the presence of Ser‐221. The residual activity of the N155G:S221A enzyme (∼104‐fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. the mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor. These data suggest that Ser‐221 functions together with Asn‐155 to accelerate amide bond hydrolysis and that other transition state stabilizing interactions account for the residual rate enhancement of 103− to 104−fold. More generally, these studies illustrate the limitations of using site‐directed mutagenesis to probe the energetic importance of a single catalytic

ISSN:0887-3585    

DOI:10.1002/prot.340070405

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe X‐ray structure of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26–Cys232 and Cys29–Cys87 and Cys22–Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as α‐helices and β‐sheets indicated systematic long‐range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that bord

ISSN:0887-3585    

DOI:10.1002/prot.340070406

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

年代:1990

数据来源: WILEY

摘要:

AbstractCrystal have been grown of myoglobin produced inEscherichia colifrom a synthetic gene, and the structure has been solved to 1.9 Å resolution. The space group of the crystals is P6, which is different from previously solved myoglobin crystal forms. The synthetic myoglobin is essentially identical to myoglobin isolated from sperm whale tissue, except for the retention of the initiator methionine at the N‐terminus and the substitution of asparagine for aspartic acid at position 122. Superposition of the coordinates of native and synthetic sperm whale myoglobins reveals only minor changes in the positions of main chain atoms and roeientation of some surface side chains. Crystals of variant of the “synthetic” myoglobin have also been grown for structural analysis of the role of key amino acid residues in ligand and speci

ISSN:0887-3585    

DOI:10.1002/prot.340070407

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

年代:1990

数据来源: WILEY

摘要:

AbstractA procedure for the construction of complete protein structures from only αcarbon coordinates is described. This involves building the backbone by sequential addition of Pro, Gly, or Ala residues. This main chain structure is then refined using molecular dynamics. Side chains are constructed by sequential addition of atoms with intermediate molecular dynamics refinement. For α lytic protease (a structure that is mostly β sheet) a backbone root mean square deviation (RMSD) of 0.19 Å and an overall RMSD of 1.24 Å from the crystallographic coordinates are attained. For troponin C (67% β‐helix), where the coordinates are available only for the α‐carbons, a backbone RMSD of 0.41 Å and an overall RMSD of 1.68 Å are attained (fits kindly provided by Dr. Michael James and Natalie Strynadka). For flavodoxin a backbone RMSD of 0.49 Å and an overall RMSD of 1.64 Å

ISSN:0887-3585    

DOI:10.1002/prot.340070408

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

年代:1990

数据来源: WILEY

摘要:

AbstractWe address herein the problem of delineating the relationships between the known protein structures. In order to study this problem, methods have been developed to represent arbitrarily sized fragments of biopolymer backbone, and to compare distributions of such fragments. These methods are applied to a classification of 123 structures representing the entire set of known x‐ray structures. The resulting data are analyzed (on the four‐Cαlength scale) to determine both the large‐scale organization of the set of known structures (i.e., the relationships between large groups of structures, each comprised of proteins that are structurally related) and its local structure (i.e., the quantitative degree of similarity between any two specific structures). It is shown that the set of structures from a continuum of structural types, ranging from allhelical to all‐sheet/barrel proteins. It is further demonstrated that the density of protein structures is not uniform across this continuum, but rather that structures cluster in certain regions, separated by regions of lower population. The properties of the various regions of the structural space are determined. The existence is demonstrated of strong quantitative correlations between the contents of different types of four‐Cαfragments within protein structures, which imply significant constraints on the types of architecture that can occur in proteins. Analysis of the distribution of structures demonstrates some hitherto unsuspected similarities and suggests that, in some circumstances, neither structural similarity no sequence homology may be necessary conditions for evolutionary relationship between proteins. It is also suggested that these unsuspected similarities may imply similar folding mechanisms for structures of apparently different global architecture. Cases are also noted in which apparently similar structures may fold by different mechanisms. The connection between structure and dynamic properties is discussed, and a possible role of dynamics in the evolution of protein structures is suggested. The sensitivity of the methods presented herein to anomalies of structure refinement is demonstrated. It is suggested that present results provide a frame‐work for analyzing experimental results on structural similarity obtained using vibrational circular dichroism spectra, which are sensitive to local backb

ISSN:0887-3585    

DOI:10.1002/prot.340070409

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

年代:1990

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340070401

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

年代:1990

数据来源: WILEY