Insights into the intramolecular coupling between the N- and C-domains of troponin C derived from high-pressure, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering studies.

Troponin C (TnC), the Ca(2+)-binding component of the troponin complex of vertebrate skeletal muscle, consists of two structurally homologous domains, the N- and C-domains; these domains are connected by an exposed α-helix. Mutants of full-length TnC and of its isolated domains have been constructed using site-directed mutagenesis to replace different Phe residues with Trp. Previous studies utilizing these mutants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and that the N-domain has no effect on the stability of the C-domain [Rocha, C. B., Suarez, M. C., Yu, A., Ballard, L., Sorenson, M. M., Foguel, D., and Silva, J. L. (2008) Biochemistry 47, 5047-5058]. Here, we analyzed the stability of full-length F29W TnC using structural approaches under conditions of added urea and hydrostatic pressure denaturation; F29W TnC is a fluorescent mutant, in which Phe 29, located in the N-domain, was replaced with Trp. From these experiments, we calculated the thermodynamic parameters (ΔV and ΔG°(atm)) that govern the folding of the intact F29W TnC in the absence or presence of Ca(2+). We found that the C-domain has only a small effect on the structure of the N-domain in the absence of Ca(2+). However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca(2+)-bound state (i.e., when Ca(2+) was also bound to sites III and IV of the C-domain). An accompanying decrease in the thermodynamic stability of the N-domain generated a reduction in ΔΔG°(atm) in absolute terms, and Ca(2+) binding affects the Ca(2+) affinity of the N-domain in full-length TnC. Cross-talk between the C- and N-domains may be mediated by the central helix, which has a smaller volume and likely greater rigidity and stability following binding of Ca(2+) to the EF-hand sites, as determined by our construction of low-resolution three-dimensional models from the small-angle X-ray scattering data.

A fluorescent mutant of the NM domain of the yeast prion Sup35 provides insight into fibril formation and stability.

The Sup35 protein of Saccharomyces cerevisiae forms a prion that generates the [PSI(+)] phenotype. Its NM region governs prion status, forming self-seeding amyloid fibers in vivo and in vitro. A tryptophan mutant of Sup35 (NM(F117W)) was used to probe its aggregation. Four indicators of aggregation, Trp 117 maximum emission, Trp polarization, thio-T binding, and light scattering increase, revealed faster aggregation at 4 degrees C than at 25 degrees C, and all indicators changed in a concerted fashion at the former temperature. Curiously, at 25 degrees C the changes were not synchronized; the first two indicators, which reflect nucleation, changed more quickly than the last two, which reflect fibril formation. These results suggest that nucleation is insensitive to temperature, whereas fibril extension is temperature dependent. As expected, aggregation is accelerated when a small fraction (5%) of the nuclei produced at 4 or 25 degrees C are added to a suspension containing the soluble NM domain, although these nuclei do not seem to propagate any structural information to the growing fibrils. Fibrils grown at 4 degrees C were less stable in GdmCl than those grown at higher temperature. However, they were both resistant to high pressure; in fact, both sets of fibrils responded to high pressure by adopting an altered conformation with a higher capacity for thio-T binding. From these data, we calculated the change in volume and free energy associated with this conformational change. AFM revealed that the fibrils grown at 4 degrees C were statistically smaller than those grown at 25 degrees C. In conclusion, the introduction of Trp 117 allowed us to more carefully dissect the effects of temperature on the aggregation of the Sup35 NM domain.

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

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.