Thermal Stability and Domain-Domain Interactions in Natural and Recombinant Protein C
| Autor: | Henryk Lubon, Carolyn L. Orthner, William N. Drohan, Leonid Medved, Timothy K. Lee, Kenneth C. Ingham |
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| Rok vydání: | 1995 |
| Předmět: |
Isothermal microcalorimetry
Protein Denaturation Hot Temperature Protein Conformation Osteocalcin Biochemistry Fluorescence law.invention law medicine Humans Denaturation (biochemistry) Thermal stability Molecular Biology Gla domain Serine protease Epidermal Growth Factor biology Transition (genetics) Chemistry Cell Biology Recombinant Proteins Crystallography Recombinant DNA biology.protein Calcium Colorimetry Protein C medicine.drug |
| Zdroj: | Journal of Biological Chemistry. 270:13652-13659 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.270.23.13652 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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