A WEAK CALCIUM-BINDING SITE IN SUBTILISIN BPN' HAS A DRAMATIC EFFECT ON PROTEIN STABILITY

The crystal structures of both subtilisin 8397 and a thermostable vari ant (Lys 256 Tyr) have been determined to 2.2 and 1.8 Angstrom resolut ion. 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 m ajor changes in the protein structure (total rms deviation is 0.41 Ang strom); 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, 83 11-8317). Unexpectedly, the weak calcium binding site was occupied in the 8397+1 structure but not in the 8397 structure. The goal of the Ly s 256 Tyr mutation was to improve the stability of subtilisin in DMF b y removing a surface charge. However, changing this residue altered ca lcium binding at a site 12 Angstrom away, illustrating the importance of structure determination in understanding stability changes.