Deciphering the role of the electrostatic interactions in the alpha-tropomyosin head-to-tail complex.
Skeletal alpha-tropomyosin (Tm) is a dimeric coiled-coil protein that forms linear assemblies under low ionic strength conditions in vitro through head-to-tail interactions. A previously published NMR structure of the Tm head-to-tail complex revealed that it is formed by the insertion of the N-terminal coiled-coil of one molecule into a cleft formed by the separation of the helices at the C-terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double-mutant Tm fragments in which specific charged residues were changed to alanine in head-to-tail binding assays, and the effects of the mutations were analyzed by thermodynamic double-mutant cycles and protein-protein docking. 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 head-to-tail complex stability by modulating the stability of the helices at the Tm termini.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Fernando
F
Corrêa
Roberto Kopke
RK
Salinas
Alexandre M J J
AM
Bonvin
Chuck S
CS
Farah
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013499:Surface Properties; D013696:Temperature; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry*
D050505:Mutant Proteins; D014335:Tropomyosin