Crystal Structure Analysis of Subtilisin BPN’ Mutants Engineered for Studying Thermal Stability
- 1 January 1996
- book chapter
- Published by Springer Nature
- Vol. 379, 159-169
- https://doi.org/10.1007/978-1-4613-0319-0_18
Abstract
The high resolution crystal structures of four genetically engineered subtilisin BPN’ variants (E.C. 3.4.21.14) which vary dramatically in their stability have been determined. The simplest variant, S3, contains two altered residues, N218S and S221C. The N218S change was incorporated for its stabilizing effects and its influence on crystallization; the S221C change, a modification of the active site serine, was included to reduce autolysis. The second variant, S12, includes the two additional stabilizing mutations M50F and Y217K. S15, the third variant, in addition to the 4 single site mutations, has residues 75–83, the high-affinity calcium-binding site, deleted. The final variant S22 incorporates all of the above changes and two additional site specific mutations, T22C and S87C, which form a stabilizing disulfide bridge. The structural changes and influence on stability of each of these mutations are discussed in the context of supporting biophysical studies.Keywords
This publication has 29 references indexed in Scilit:
- Energetics of folding subtilisin BPN'Biochemistry, 1992
- Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solutionBiochemistry, 1991
- Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfoldingBiochemistry, 1989
- The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test caseBiochemistry, 1988
- Calculation of an OMIT mapJournal of Applied Crystallography, 1988
- The use of an imaging proportional counter in macromolecular crystallographyJournal of Applied Crystallography, 1987
- Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bondBiochemistry, 1987
- Complete primary structure of thermitase from Thermoactinomyces vulgaris and its structural features related to the subtilisin‐type proteinasesFEBS Letters, 1985
- The protein data bank: A computer-based archival file for macromolecular structuresJournal of Molecular Biology, 1977
- A comparison of crystallographic data of the subtilopeptidases B and CJournal of Molecular Biology, 1967