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. Study holds ProTherm entries: 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191 Extra Details: additive : EDTA(2 mM), recombinant protein C; serine protease; two domains;,epidermal growth factor-like; structural changes

Submission Details

ID: sacGEmr4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Medved LV;Orthner CL;Lubon H;Lee TK;Drohan WN;Ingham KC,J. Biol. Chem. (1995) Thermal stability and domain-domain interactions in natural and recombinant protein C. PMID:7775416
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3JTC 2011-04-06 1.6 Importance of Mg2+ in the Ca2+-Dependent Folding of the gamma-Carboxyglutamic Acid Domains of Vitamin K-Dependent clotting and anticlotting Proteins
1LQV 2002-06-19 1.6 Crystal structure of the Endothelial protein C receptor with phospholipid in the groove in complex with Gla domain of protein C.
4DT7 2012-05-09 1.9 Crystal structure of thrombin bound to the activation domain QEDQVDPRLIDGKMTRRGDS of protein C
3F6U 2008-11-25 2.8 Crystal structure of human Activated Protein C (APC) complexed with PPACK
1AUT 1997-08-20 2.8 Human activated protein C

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.7 Vitamin K-dependent protein C Q28506 PROC_MACMU
200.0 C,L ACTIVATED PROTEIN C P04070 PROC_HUMAN