The Engineering of Binding Affinity at Metal Ion Binding Sites for the Stabilization of Proteins: Subtilisin as a Test Case

Michael W. Pantoliano, Marc Whitlow, Jay F. Wood, Michele L. Rollence, Barry C. Finzel, Gary L. Gilliland, Thomas L. Poulos, Philip N. Bryan

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166 Scopus citations


abstract;A weak Ca2+binding site in the bacterial serine protease subtilisin BPN′ (EC was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN’ at 65 °C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+specific binding site with an apparent log Ka= 1.49. A series of refined X-ray crystal structures (R ≤ 0.15, 1.7 Å) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2has allowed a detailed structural characterization of this Ca2+binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 Å, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory. Moreover, when both Asp changes were introduced together, the binding affinity for Ca2+was found to be increased about 6-fold over that for the wild-type protein, suggesting an independent and nearly additive effect on the total electrostatic potential at this locus.

Original languageEnglish (US)
Pages (from-to)8311-8317
Number of pages7
Issue number22
StatePublished - Oct 1 1988

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