title: The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.
abstract: In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas "Prof. Alejandro C. Paladini" (IQUIFIB), Universidad de Buenos Aires, Junín 956, 1113AAD Buenos Aires, Argentina.
authors.forename: Diego S
authors.initials: DS
authors.lastname: Vazquez
authors.affiliation: Laboratorio de Fisiología de Proteínas, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales and IQUIBICEN-CONICET, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina. Electronic address: nachoquique@gmail.com.
authors.forename: Ignacio E
authors.initials: IE
authors.lastname: Sánchez
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas "Prof. Alejandro C. Paladini" (IQUIFIB), Universidad de Buenos Aires, Junín 956, 1113AAD Buenos Aires, Argentina.
authors.forename: Ana
authors.initials: A
authors.lastname: Garrote
authors.affiliation: CONICET e Instituto de Energía y Desarrollo Sustentable (IEDS), Centro Atómico Bariloche, Comisión Nacional de Energía Atómica (CNEA), Av. E. Bustillo km 9.500, San Carlos de Bariloche, Río Negro, Argentina. Electronic address: mp.sica@gmail.com.
authors.forename: Mauricio P
authors.initials: MP
authors.lastname: Sica
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas "Prof. Alejandro C. Paladini" (IQUIFIB), Universidad de Buenos Aires, Junín 956, 1113AAD Buenos Aires, Argentina. Electronic address: jsantos@qb.ffyb.uba.ar.
authors.forename: Javier
authors.initials: J
authors.lastname: Santos
mesh_terms: D004926:Escherichia coli; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
chemical_list: D013879:Thioredoxins
keywords: Folding kinetics; Intermediate state; Molecular dynamics; Protein stability; Transition state ensemble