PMID: 2504281May 30, 1989Paper

Protein engineering of disulfide bonds in subtilisin BPN'

Biochemistry
C Mitchinson, J A Wells

Abstract

Five single-disulfide mutants were studied in subtilisin BPN', a cysteine-free, secreted serine protease from Bacillus amyloliquefaciens. The disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. These bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 A from the catalytic Ser-221, and linked residues that were separated by 39 up to 206 amino acids. All disulfide bonds formed in the enzyme when the expressed protein was secreted from Bacillus subtilis, and the disulfides had only minor effects on the enzyme kinetics. Although these disulfide bonds varied by over 50-fold in their equilibrium constants for reduction with dithiothreitol, there was no correlation between the strength of the disulfide bond and the stability it imparted to the enzyme to irreversible inactivation. In some cases, the disulfide-bonded protein was stabilized greatly relative to its reduced counterpart. However, no disulfide mutant was substantially more stable than wild-type subtilisin BPN'. Some of these results can be rationalized by destabilizing effects of the cysteine mutations that disrupt interactions present in the folded enzyme structure. It is also pos...Continue Reading

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Nov 1, 1979·Analytical Biochemistry·E G DelMarM C Geokas
Apr 18, 1978·Biochemistry·R E JohnsonJ A Rupley
Jun 25, 1977·Journal of Molecular Biology·T E Creighton
Dec 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·A A PakulaR T Sauer
Jan 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·R WetzelW J Becktel
Apr 21, 1987·Biochemistry·J E VillafrancaJ Kraut
Apr 15, 1988·Science·T E Creighton
Apr 5, 1986·Journal of Molecular Biology·C N Pace, T E Creighton
Oct 7, 1986·Biochemistry·C O Pabo, E G Suchanek
Oct 14, 1970·Journal of Molecular Biology·M Mandel, A Higa
Mar 27, 1972·European Journal of Biochemistry·J DrenthR Koekoek
Feb 14, 1971·Journal of Molecular Biology·B Lee, F M Richards
Jan 18, 1969·Nature·C S WrightJ Kraut
Jan 1, 1983·Journal of Bacteriology·S Pollitt, H Zalkin
Nov 18, 1983·Science·J E VillafrancaJ Kraut
Apr 25, 1982·Journal of Molecular Biology·Y Goto, K Hamaguchi
Jan 1, 1981·Advances in Protein Chemistry·J S Richardson
May 1, 1961·Journal of Bacteriology·C Anagnostopoulos, J Spizizen

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Citations

Oct 1, 1993·Protein Science : a Publication of the Protein Society·S F Betz
Mar 1, 1997·Protein Science : a Publication of the Protein Society·R J Siezen, J A Leunissen
Apr 1, 1993·Journal of Protein Chemistry·Y KobayashiN Saitô
Aug 1, 1993·Journal of Computer-aided Molecular Design·G Vriend, V Eijsink
Oct 1, 1995·Molecular Biotechnology·P W Goodenough
Mar 1, 1993·The International Journal of Biochemistry·H Takagi
Sep 21, 1994·Biochimica Et Biophysica Acta·H TatsumiH Motai
Sep 27, 1995·Biochimica Et Biophysica Acta·C O Fágáin
Mar 1, 1993·Journal of Biotechnology·D W GoddetteC R Wilson
Jan 1, 1991·Biotechnology Advances·C O Fágáin, R O'Kennedy
Aug 1, 1991·Current Opinion in Biotechnology·A C Storer
Feb 18, 2004·Protein Expression and Purification·Ka M Ho, Boon L Lim
Jul 30, 2002·Journal of Molecular Biology·Daisy SahooVasanthy Narayanaswami
Mar 12, 2003·Journal of Molecular Biology·John W Blankenship, Philip E Dawson
Jan 11, 2001·Biochimica Et Biophysica Acta·P N Bryan
Oct 11, 2003·Biotechnology Advances·C G Kumar, H Takagi
Aug 17, 1999·Current Opinion in Biotechnology·A ShawA G Day
Jul 2, 2010·Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology·Mehdi ImaniMahboobeh Nazari
Dec 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·T Y Lin, P S Kim
Apr 22, 2003·Annual Review of Biochemistry·Daniel A KrautDaniel Herschlag
Aug 13, 2013·PloS One·Inmaculada Sanchez-RomeroJose M Sanchez-Ruiz
Aug 18, 1999·Proceedings of the National Academy of Sciences of the United States of America·S Atwell, J A Wells
Jan 1, 1990·Trends in Biochemical Sciences·C N Pace
Jan 16, 2013·FEBS Letters·Qing LiBrent L Iverson
Jan 22, 2009·Biophysical Chemistry·Pascal Pecher, Ulrich Arnold
Jun 30, 2006·Proteins·Jean-Luc Pellequer, Shu-wen W Chen
Jun 16, 2011·Protein Science : a Publication of the Protein Society·Witold A Witkowski, Jeanne A Hardy
Mar 27, 2016·Journal of Molecular Biology·Nicholas J FraserColin J Jackson
Nov 20, 1992·Journal of Molecular Biology·D W GoddetteR J Fletterick
Jan 13, 2015·New Biotechnology·Ljubica VojcicUlrich Schwaneberg
Oct 3, 2002·Journal of Biochemistry and Molecular Biology·Hyeung Jin JangYu Sam Kim

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