title: Hydrophobic repacking of the dimer interface of triosephosphate isomerase by in silico design and directed evolution.
abstract: Triosephosphate isomerase from Saccharomyces cerevisiae (wt-TIM) is an obligated homodimer. The interface of wt-TIM is formed by 34 residues. In the native dimer, each monomer buries nearly 2600 A(2) of accessible surface area (ASA), and 58.4% of the interface ASA is hydrophobic. We determined the thermodynamic and functional consequences of increasing the hydrophobic character of the wt-TIM interface. Mutations were restricted to a cluster of five nonconserved residues located far from the active site. Two different approaches, in silico design and directed evolution, were employed. In both methodologies, the obtained proteins were soluble, dimeric, and compact. In silico-designed proteins are very stable dimers that bind substrate with a wild-type-like K(m); albeit, they exhibited a very low k cat. Proteins obtained from directed evolution experiments show wild-type-like catalytic activity, while their stability is decreased. Hydrophobic replacements at the interface produced a remarkable shift in the dissociation step. For wt-TIM and for TIMs obtained by directed evolution, dissociation was observed in the first transition, with C(1/2) values ranging from 0.58 to 0.024 M GdnHCl, whereas for TIMs generated by in silico design, dissociation occurred in the last transition, with C(1/2) values ranging form 3.01 to 3.65 M GdnHCl. For the latter mutants, the stabilization of the interface changed the equilibrium transitions to a novel four-state process with two dimeric intermediates. The change in the intermediate nature suggests that the relative stabilities of different folding units are similar so that subtle alterations in their stability produce a total transformation of the folding pathway.
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. Postal 70-159, 04510 México, DF, Mexico.
authors.forename: Mariana
authors.initials: M
authors.lastname: Peimbert
authors.forename: Lenin
authors.initials: L
authors.lastname: Domínguez-Ramírez
authors.forename: D Alejandro
authors.initials: DA
authors.lastname: Fernández-Velasco
mesh_terms: D002384:Catalysis; D003198:Computer Simulation; D019281:Dimerization; D019020:Directed Molecular Evolution; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014305:Triose-Phosphate Isomerase