Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C

Troponin C (TnC) is the Ca2+-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed (x-helix. The C-domain has two high-affinity sites for Ca2+ that also bind Mg2+, whereas the N-domain has two low-affinity sites for Ca2+. Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/ N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca2+. Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca2+ binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca2+ affinity of the N-domain in solution. From the In K-obs versus ln alpha(H2O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N double left right arrow N.Ca-2 that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 Angstrom(2). In skinned fibers, the affinity for Ca2+ was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca2+ are critically dependent on protein hydration.