Equilibrium unfolding and conformational plasticity of troponin I and T.
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.
Martins
Samantha M
SM
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil.
Chapeaurouge
Alex
A
Ferreira
Sérgio T
ST
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry*; D020107:Troponin T / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D019210:Troponin I; D020107:Troponin T; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014508:Urea; D019791:Guanidine