THERMAL STABILITY AND DOMAIN-DOMAIN INTERACTIONS IN NATURAL AND RECOMBINANT PROTEIN C

Abstract Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a T of 62°C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20°C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa -carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF) fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca. In 2 mM Ca, the first heat absorption peak in both intact proteins became biphasic, indicating Ca-induced structural changes. By contrast, Ca had very little effect on the melting of Gla-domainless protein C. This indicates that not Ca itself but the Ca-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca and EDTA suggests that Ca induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.