Different effects of trifluoroethanol and glycerol on the stability of tropomyosin helices and the head-to-tail complex.
Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 A), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.
Corrêa
Fernando
F
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Farah
Chuck S
CS
D001665:Binding Sites; D003198:Computer Simulation; D004355:Drug Stability; D005990:Glycerol / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D014270:Trifluoroethanol / Q000737:chemistry*; D014335:Tropomyosin / Q000737:chemistry* / Q000648:ultrastructure*
D014335:Tropomyosin; D014270:Trifluoroethanol; D005990:Glycerol