A weak calcium binding site in subtilisin BPN′ has a dramatic effect on protein stability

Richard D. Kidd, Hemant P. Yennawar, Pamela Sears, Chi Huey Wong, Gregory K. Farber

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The crystal structures of both subtilisin 8397 and a thermostable variant (Lys 256 Tyr) have been determined to 2.2 and 1.8 Å resolution. The thermostable variant (8397+1) was previously shown to exhibit enhanced thermostability over 8397 in both aqueous solutions and the polar organic solvent dimethylformamide (Sears, P.; et al. J. Am. Chem. Soc. 1994, 116, 6521-6530). The single substitution did not induce major changes in the protein structure (total rms deviation is 0.41 Å); however, changes in calcium binding were detected. The strong calcium binding site was occupied in both structures as has been seen in other subtilisins (Pantoliano, M.; et al. Biochemistry 1988, 27, 8311-8317). Unexpectedly, the weak calcium binding site was occupied in the 8397+1 structure but not in the 8397 structure. The goal of the Lys 256 Tyr mutation was to improve the stability of subtilisin in DMF by removing a surface charge. However, changing this residue altered calcium binding at a site 12 Å away, illustrating the importance of structure determination in understanding stability changes.

Original languageEnglish (US)
Pages (from-to)1645-1650
Number of pages6
JournalJournal of the American Chemical Society
Volume118
Issue number7
DOIs
StatePublished - Feb 21 1996

Fingerprint

Subtilisins
Protein Stability
Binding sites
Calcium
Binding Sites
Proteins
Subtilisin
Dimethylformamide
Biochemistry
Surface charge
Organic solvents
Substitution reactions
Crystal structure
Mutation

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Kidd, Richard D. ; Yennawar, Hemant P. ; Sears, Pamela ; Wong, Chi Huey ; Farber, Gregory K. / A weak calcium binding site in subtilisin BPN′ has a dramatic effect on protein stability. In: Journal of the American Chemical Society. 1996 ; Vol. 118, No. 7. pp. 1645-1650.
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abstract = "The crystal structures of both subtilisin 8397 and a thermostable variant (Lys 256 Tyr) have been determined to 2.2 and 1.8 {\AA} resolution. The thermostable variant (8397+1) was previously shown to exhibit enhanced thermostability over 8397 in both aqueous solutions and the polar organic solvent dimethylformamide (Sears, P.; et al. J. Am. Chem. Soc. 1994, 116, 6521-6530). The single substitution did not induce major changes in the protein structure (total rms deviation is 0.41 {\AA}); however, changes in calcium binding were detected. The strong calcium binding site was occupied in both structures as has been seen in other subtilisins (Pantoliano, M.; et al. Biochemistry 1988, 27, 8311-8317). Unexpectedly, the weak calcium binding site was occupied in the 8397+1 structure but not in the 8397 structure. The goal of the Lys 256 Tyr mutation was to improve the stability of subtilisin in DMF by removing a surface charge. However, changing this residue altered calcium binding at a site 12 {\AA} away, illustrating the importance of structure determination in understanding stability changes.",
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A weak calcium binding site in subtilisin BPN′ has a dramatic effect on protein stability. / Kidd, Richard D.; Yennawar, Hemant P.; Sears, Pamela; Wong, Chi Huey; Farber, Gregory K.

In: Journal of the American Chemical Society, Vol. 118, No. 7, 21.02.1996, p. 1645-1650.

Research output: Contribution to journalArticle

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