Free-energy linkage between folding and calcium binding in EF-hand proteins

Abstract

Troponin is the singular Ca2+-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca 2+-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca2+ was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (ΔV and ΔGatm). In this study, we propose that part of the affinity for Ca2+ is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca2+ binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca2+ in EF-hand proteins, which is crucial to function. © 2008 by the Biophysical Society.