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

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39 Scopus citations


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
Issue number7
StatePublished - Feb 21 1996

All Science Journal Classification (ASJC) codes

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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