title: Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes.
abstract: The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins.
authors.affiliation: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, 04510 México DF, México.
authors.forename: Monica
authors.initials: M
authors.lastname: Rodríguez-Bolaños
authors.affiliation: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, 04510 México DF, México.
authors.forename: Nallely
authors.initials: N
authors.lastname: Cabrera
authors.affiliation: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, 04510 México DF, México ruy@ifc.unam.mx.
authors.forename: Ruy
authors.identifier: 0000-0003-0810-1159
authors.initials: R
authors.lastname: Perez-Montfort
mesh_terms: D020816:Amino Acid Motifs; D002942:Circular Dichroism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D015800:Protozoan Proteins; D014305:Triose-Phosphate Isomerase
keywords: critical residues; guanidine hydrochloride; protein folding; reactivation; site-directed mutagenesis; triosephosphate isomerase