title: 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 Tm of approximately 62 degrees 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 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-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 Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.
authors.affiliation: J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
authors.forename: L V
authors.initials: LV
authors.lastname: Medved
authors.forename: C L
authors.initials: CL
authors.lastname: Orthner
authors.forename: H
authors.initials: H
authors.lastname: Lubon
authors.forename: T K
authors.initials: TK
authors.lastname: Lee
authors.forename: W N
authors.initials: WN
authors.lastname: Drohan
authors.forename: K C
authors.initials: KC
authors.lastname: Ingham
mesh_terms: D002118:Calcium / Q000494:pharmacology; D003124:Colorimetry; D004815:Epidermal Growth Factor / Q000737:chemistry; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D015675:Osteocalcin / Q000737:chemistry; D011486:Protein C / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011486:Protein C; D011994:Recombinant Proteins; D015675:Osteocalcin; D004815:Epidermal Growth Factor; D002118:Calcium