Volume and Free Energy of Folding for Troponin C C-Domain: Linkage to Ion Binding and N-Domain Interaction

Abstract

Troponin C (TnC) is an 18-kDa acidic protein of the EF-hand family that serves as the trigger for muscle contraction. In this study, we investigated the thermodynamic stability of the C-domain of TnC in all its occupancy states (apo, Mg (2+)-, and Ca (2+)-bound states) using a fluorescent mutant with Phe 105 replaced by Trp (F105W/C-domain, residues 88-162) and (1)H NMR spectroscopy. High hydrostatic pressure was employed as a perturbing agent, in combination with urea or without it. On the basis of changes in Trp emission, the C-domain apo state was denatured by pressure (in the range of 1-1000 bar) in the absence of urea. The fluorescence data were corroborated by following the changes in the (1)H NMR signal of Histidine 128. Addition of Ca (2+) or Mg (2+) increased the C-domain stability so that complete denaturation was attained only by the combined use of high hydrostatic pressure and either 7-8 M or 1.5-2 M urea, respectively. The (1)H NMR spectra in the presence of Ca (2+) was typical of a highly structured protein and allowed us to follow the changes in the local environment of several amino-acid residues as a function of pressure at 4 M Urea. Different residues presented different volume changes, but those that are in the hydrophobic core portrayed values very similar to that obtained for tryptophan 105 as measured by fluorescence, indicating that it is indeed a good probe for the overall tertiary structure. From these experiments, we calculated the thermodynamic parameters (Delta G degrees atm and Delta V) that govern the folding of the C-domain in all its possible physiological states and constructed a thermodynamic cycle. Furthermore, a comparison of the volume and free-energy changes of folding of isolated C-domain with those of intact TnC (F105W) revealed that the N-domain has little effect on the structure of the C-domain, even in the presence of Ca (2+). The volume and free-energy diagrams reveal a landscape of different conformations from the less structured, denatured apo form to the highly structured, Ca (2+)-bound form. The large change in folding free energy of the C-domain that takes place when Ca (2+) binds may explain the much higher Ca (2+) affinity of sites III and IV, 2 orders of magnitude higher than the affinity of sites I and II.

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

“…Because the high-pressure NMR cell can withstand up to 5 kbar (27), it is possible to derive the values of volume change in the absence of urea for the conditions without Ca 21 . As observed for the C-domain of TnC (14), the largest magnitudes for DV were associated with the amino acid residues that are located in the hydrophobic core of the protein. The deviations in DV along the protein demonstrate how unfolding affects protein structure in a site-specific manner, which noticeably depends on the stabilizing forces in the local environment of each residue.…”

“…Strikingly, the center of spectral mass of the Ca 21 -bound form did not change even at high concentrations of urea. These data show that the structure of the F29W/N-domain is remarkably stable when Ca 21 is bound, as seen with the C-domain (14). The unfolding process is better visualized when the degree of denaturation (a) is plotted as a function of urea concentration (Fig.…”

“…In a previous study (14), we used F105W/C-domain (residues 88-162) and F105W (residues 1-162) to address questions related to the stability of the C-domain, using W105 (located in site III) as a probe. Monitored by changes in Trp emission, the C-domain apo state was denatured by pressure (in the range of 1-1000 bar) in the absence of urea.…”

“…Additionally, by performing pressure titration at 1°C, we evaluated the effects of low temperature on the thermodynamic stability of the F29W/N-domain, because the N-domain has a higher affinity for Ca 21 in this condition (15). In combination with our previous article (14) in which denaturation of the C-domain was investigated, we observed that the increase in stability of the C-domain when Ca 21 binds is .50% greater than that caused by Ca 21 binding to the N-domain. However, the sites in the C-domain (III and IV) have a higher affinity for Ca 21 than the sites in the N-domain (I and II).…”

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.…”