Thermodynamic and structural studies of the head-to-tail complex of the muscular alpha-Tropomyosin

Author(s)

Fernando Corrêa

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2008-06-20

Examining board members

Shaker Chuck Farah; Ana Paula Valente Lacerda de Almeida; Richard Charles Garratt; Hernan Chaimovich Guralnik; Sandro Roberto Marana

• Advisor: Shaker Chuck Farah

Abstract

Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation. (AU)