title: The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.
abstract: The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β-turn occurrence, we engineered two chimerical enzymes where their super secondary β-loop-α motifs 2 ((βα)2) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα)2motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)2motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα)2of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (EA). A systematic analysis of DSC data showed a large decrease on the EAof TcTIM (ΔEAranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Laboratorio de Biofisicoquímica, Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, CdMx, 04510, México.
authors.forename: Valeria
authors.initials: V
authors.lastname: Guzmán-Luna
authors.affiliation: Laboratorio de Biofisicoquímica, Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, CdMx, 04510, México.
authors.forename: Andrea G
authors.initials: AG
authors.lastname: Quezada
authors.affiliation: Laboratorio de Biofisicoquímica, Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, CdMx, 04510, México.
authors.forename: A Jessica
authors.initials: AJ
authors.lastname: Díaz-Salazar
authors.affiliation: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, CdMx, 04510, 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, CdMx, 04510, México.
authors.forename: Ruy
authors.initials: R
authors.lastname: Pérez-Montfort
authors.affiliation: Laboratorio de Biofisicoquímica, Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, CdMx, 04510, México.
authors.forename: Miguel
authors.initials: M
authors.lastname: Costas
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005986:Glyceraldehyde 3-Phosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D015202:Protein Engineering; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D005986:Glyceraldehyde 3-Phosphate; D011392:Proline; D014305:Triose-Phosphate Isomerase
keywords: TIM barrels; activation energy; beta-loop-alpha motifs; beta-turns; contact maps; differential scanning calorimetry; parasites; protein engineering; thermal unfolding