Free-Energy Linkage between Folding and Calcium Binding in EF-Hand Proteins

Abstract

Troponin is the singular Ca(2+)-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 Ca(2+) was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (DeltaV and DeltaG(atm)). In this study, we propose that part of the affinity for Ca(2+) is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca(2+) 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 Ca(2+) in EF-hand proteins, which is crucial to function.

Auto-inhibitory role of the EF-SAM domain of STIM proteins in store-operated calcium entry

“…S2) (23); how- ever, STIM2 EF-SAM is considerably more stable than STIM1. Analogous to our STIM observations, the C-terminal EF-hand domain of TnC has a lower stability than the N-terminal domain, despite a higher Ca 2þ affinity; further, the elevated affinity is attributable to the greater degree of folding (i.e., larger energetic barrier to Ca 2þ -induced unfolding) which occurs upon Ca 2þ binding compared to the N-terminal domain (33). A similar phenomenon occurs in the STIM proteins, where the more stable apo EF-SAM and lesser Ca 2þ -induced folding of the STIM2 EF-hand contributes to a lower affinity.…”

“…The Ca 2þ -binding induced stabilization of EF-hands is a well conserved feature of these common protein domains. For example, the isolated C-and N-terminal EF-hand domains of Troponin C (TnC), each undergo considerable stabilization upon Ca 2þ -binding (33). Despite the high sequence and structural homology to STIM1, the STIM2 EF-hand binds Ca 2þ with lower apparent affinity than the STIM1 counterpart (Fig.…”

A hypothesis to reconcile the physical and chemical unfolding of proteins

“…In the case of apomyoglobin, although separate elegant works on pressure denaturation (56) and urea denaturation (57) exist, NMR data have been extensively obtained at 8 M urea and not at intermediate urea concentrations. Considerable fluorescence and NMR data are available for the urea and pressure denaturation of the N and C domains of troponin C (24,(58)(59)(60). Clearly, MpNep2 is highly sensitive to urea and pressure.…”

Single-Molecule Folding Mechanism of an EF-Hand Neuronal Calcium Sensor

“…These proteins relay cellular changes of Ca 2+ through intra-or interdomain rearrangements that allow them to interact with binding partners. Despite detailed structure characterization and elucidation of switch mechanisms in the native state (Capozzi et al, 2006;Grabarek, 2006), the folding mechanism of this large family of proteins is widely unexplored, with only a subset of systems studied (Aravind et al, 2008;Mukherjee et al, 2007;Stigler et al, 2011;Suarez et al, 2008;Yamniuk et al, 2007). To rationalize the conformational response of sensory EF-hand proteins to changes in Ca 2+ concentration and its relation to function, the details of their folding mechanism can prove essential.…”