Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw, William V.

doi:10.1042/bj2460001

Cited by 78 publications

(18 citation statements)

References 127 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Site-directed mutagenesis is an important tool in probing the structural and functional significance of particular residues within a protein sequence (for reviews, see Knowles, 1987;Shaw, 1987). Amino acid residues might be altered to check for their participation in catalysis, cofactor or substrate binding, molecular and receptor recognition, domain interfaces, oligomeric interactions, and the like.…”

Section: Resultsmentioning

confidence: 99%

“…In the review by Shaw (1987) on specific point mutations for several molecular species, the GlyAla substitution is one of the most frequent mentioned. Apparently the proteins maintained their fold while proven assays displayed altered activity.…”

Section: Resultsmentioning

confidence: 99%

“…Site-directed mutagenesis has become a very important and yet facile tool to explore the structural and functional significance of particular residues within proteins (for example, see Knowles, 1987;Shaw, 1987;Gruetter et al, 1987). A typical experiment would involve substitutions of an amino acid thought to be essential for catalysis and then assaying the resultant variant for activity.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Suggestions for “safe” residue substitutions in site-directed mutagenesis

Bordo

Argos

1991

Journal of Molecular Biology

366

246

View full text Add to dashboard Cite

The conserved topological structure observed in various molecular families such as globins or cytochromes c allows structural equivalencing of residues in every homologous structure and defines in a coherent way a global alignment in each sequence family. A search was performed for equivalent residue pairs in various topological families that were buried in protein cores or exposed at the protein surface and that had mutated but maintained similar unmutated environments. Amino acid residues with atoms in contact with the mutated residue pairs defined the environment. Matrices of preferred amino acid exchanges were then constructed and preferred or avoided amino acid substitutions deduced. Given the conserved atomic neighborhoods, such natural in vivo substitutions are subject to similar constraints as point mutations performed in site-directed mutagenesis experiments. The exchange matrices should provide guidelines for "safe" amino acid substitutions least likely to disturb the protein structure, either locally or in its overall folding pathway, and most likely to allow probing of the structural and functional significance of the substituted site.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Suggestions for “safe” residue substitutions in site-directed mutagenesis

Bordo

Argos

1991

Journal of Molecular Biology

366

246

View full text Add to dashboard Cite

show abstract

“…Several tentative rules for increasing the thermostability of proteins have been established (1,15,24) but precise predictions for changes of structure to function relationships remain elusive (see ref. 26 for review).…”

Section: Discussionmentioning

confidence: 99%

Hybrid bacillus endo-(1–3, 1–4)-β-glucanases: Construction of recombinant genes and molecular properties of the gene products

Borriss

Olsen

Thomsen

et al. 1989

Carlsberg Res. Commun.

View full text Add to dashboard Cite

Hybrid 13-glucanase genes were constructed by the reciprocal exchange of the two halves of the isolated fl-glucanase genes from Bacillus amyloliquefaciens and B. macerans. The I]-glucanase hybrid enzyme 1 (HI) contains the 107 amino-terminal residues of mature B. amyloliquefaciens [i-glucanase and the 107 carboxyl-terminal amino acid residues of B. macerans ~glucanase. The reciprocal ~glucanase hybrid enzyme 2 (H2) consists of the 105 amino-terminal residues from the B. macerans enzyme and the carboxyl-terminal 107 amino acids from B. amyloliquefaciens. The biochemical properties of the two hybrid enzymes differ significantly from each other as well as from both parental ~glucanases.Hybrid ~glucanase H 1 exhibits increased thermostability in comparison to other ~glucanases, especially in an acidic environment. This hybrid enzyme has maximum activity between pH 5.6 and 6.6, whereas the oH-optimum for enzymatic activity of B. amyloliquefaciens ~glucanase was found to be at pH 6 to 7 and for B. macerans at pH 6.0 to 7.5. Hybrid enzyme 1 being more heat stable than both parental enzymes represents a case of intragenic heterosis.Hybrid 13-glucanase 2 (H2) was found to be more thermolabile than the naturally occurring ~ghicanases it was derived from and the pH-optimum for enzymatic activity was determined to be between pH 7 and pH 8. INTRODUCI'IONThe mixed linked ( 1-3, l-4)q3-glucans constitute the major part of the endosperm cell walls of cereals like oat and barley. They can cause severe problems in the brewing industry such as reduced yield of extract and lowered rates of wort separation or beer filtration. Remaining fl-glucans in the finished beer may lead to formation of hazes and gelatinous precipitates ( 11 ). Barley ( I-3,1-4)-~-glucanases (EC 3.2.1.73) are synthesized in the scutellum and the aleurone layer during the early stages of germiAbbreviations: aa = amino adds; amp = ampicillin; bgl = ~-glucanase gene; ~glucan = (1-3,1-4)-~-D-glncan; PAGE = polyacrylamide gel clectrophoresis; SDS ffi sodium dodecyl sulfate; tet ffi tetracycline; SP = signal peptide.Springer-Verlag

show abstract

“…However, comparatively little is known of the detailed mechanisms involved in such macromolecular assembly [1]. A closely related problem is that of the role of disulphide bridges in the stabilization of protein conformation [2].…”

Section: Introductionmentioning

confidence: 99%

Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli

1988

View full text Add to dashboard Cite

By site-directed mutagenesis, Thr-75 was converted to Cys-75 in the glutathione reductase (EC 1.6.4.2) of Escherichia coli. This led to the spontaneous formation of an intersubunit disulphide bridge across the 2-fold axis of the dimeric enzyme. The disulphide bridge had no deleterious effect on the catalytic activity, but nor did it increase the thermal stability of the enzyme, possibly because of local conformational flexibility on the dimer interface. The T75C mutant, like the wild-type enzyme, was inactivated by NADPH, proving that this inactivation cannot be due to simple dissociation of the dimer.

show abstract

Protein engineering. The design, synthesis and characterization of factitious proteins

Cited by 78 publications

References 127 publications

Suggestions for “safe” residue substitutions in site-directed mutagenesis

Suggestions for “safe” residue substitutions in site-directed mutagenesis

Hybrid bacillus endo-(1–3, 1–4)-β-glucanases: Construction of recombinant genes and molecular properties of the gene products

Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli

Contact Info

Product

Resources

About