Equilibrium unfolding and conformational plasticity of troponin I and T

Abstract

The structures and stabilities of recombinant chicken muscle troponin I (TnI) and T (TnT) were investigated by a combination of bis-ANS binding and equilibrium unfolding studies. Unlike most folded proteins, isolated TnI and TnT bind the hydrophobic fluorescent probe bis-ANS, indicating the existence of solvent-exposed hydrophobic domains in their structures. Bis-ANS binding to binary or ternary mixtures of TnI, TnT and troponin C (TnC) in solution is significantly lower than binding to the isolated subunits, which can be explained by burial of previously exposed hydrophobic domains upon association of the subunits to form the native troponin complex. Equilibrium unfolding studies of TnT and TnI by guanidine hydrochloride and urea monitored by changes in far-UV CD and bis-ANS fluorescence revealed noncooperative folding transitions for both proteins and the existence of partially folded intermediate states.Taken together, these results indicate that isolated TnI and TnT are partially unstructured proteins, and suggest that conformational plasticity of the isolated subunits may play an important role in macromolecular recognition for the assembly of the troponin complex.

Folding Intermediates of the Prion Protein Stabilized by Hydrostatic Pressure and Low Temperature

“…Bis-ANS is an environment-sensitive fluorescent dye that normally binds to exposed hydrophobic surfaces in partially folded intermediates more tightly than to both the native and random coil states of proteins (37). Binding to exposed hydrophobic domains in proteins brings about a large increase in bis-ANS fluorescence emission and a blue shift of the emission maximum (31,32,38,39). Titration of the native prion protein (2 M) with bis-ANS (0 -12 M) showed saturation of bis-ANS binding sites at 4 M bis-ANS, suggesting that there are two bis-ANS binding sites/ molecule of prion protein (data not shown).…”

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

“…Troponin I (TnI, the inhibitory subunit) is a highly flexible protein that is able to adapt favorable conformations to interact with both TnC and TnT, as well as with actin [58]. There are three isoforms of TnI expressed in vertebrate striated muscles: the fast and slow skeletal isoforms (sTnI) and the cardiac-specific isoform [59-61].…”

An Interplay between Protein Disorder and Structure Confers the Ca2+ Regulation of Striated Muscle

“…TnI is not structured outside of the Tn complex. 16 TnT is divided into TnT-T1 and TnT-T2; T1 corresponds to the N-terminal domain.…”

“…Intriguingly, there are reports that the TnI⋅TnC interaction is stable in the presence of denaturing concentrations of urea. 16,30 The favorable contribution of unfolded states to association kinetics could account for the apparent urea stability.…”