Abstract
Introduction of a fifth carboxylate into the ligand array of the CD site (via the combined S55D and E59D mutations) or the EF site (G98D) of rat alpha-parvalbumin substantially increases divalent ion affinity. This behavior, in conflict with that seen in model peptide systems, agrees with existing data for rat beta-parvalbumin [Henzl et al. (1996) Biochemistry 35, 5856-5869]. The complete analysis of the S55D/E59D double variant necessitated characterization of alpha E59D. Whereas the D59E mutation has minimal influence on beta CD site affinity, E59D has a major impact on the alpha CD site, lowering the apparent association constant by a factor of 14. The thermodynamic consequences of exchanging the rat alpha CD and EF site ligand arrays, which differ at the +z and -x coordination positions, were also examined. When the alpha CD array is imported into the EF site, it acquires a low-affinity phenotype, in agreement with previous findings for beta [Henzl et al. (1998) Biochemistry 37, 9101-9111]. However, when the EF ligand array is introduced into the alpha CD binding loop, it retains a high-affinity signature. This result, contrary to that observed in beta, suggests that the influence of the parvalbumin CD site environment supersedes the intrinsic behavior of the ligand array, a conclusion further supported by the disparate impact of the beta D59E and alpha E59D mutations.
Published Version
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