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

被引:19
作者
MEDVED, LV
ORTHNER, CL
LUBON, H
LEE, TK
DROHAN, WN
INGHAM, KC
机构
[1] Holland Laboratory, Rockville, MD 20855
关键词
D O I
10.1074/jbc.270.23.13652
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-m of similar to 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 Ca2+-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domainless protein C. This indicates that not Ca2+ itself but the Ca2+-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.
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页码:13652 / 13659
页数:8
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