The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.
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)2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)2 motif 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)2 of 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 EA of TcTIM (ΔEA ranging 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.
['Guzmán-Luna|Valeria|V|', 'Quezada|Andrea G|AG|', 'Díaz-Salazar|A Jessica|AJ|', 'Cabrera|Nallely|N|', 'Pérez-Montfort|Ruy|R|', 'Costas|Miguel|M|']
D000595:Amino Acid Sequence D001665:Binding Sites D003001:Cloning, Molecular D004795:Enzyme Stability D004926:Escherichia coli D015870:Gene Expression D005986:Glyceraldehyde 3-Phosphate D007700:Kinetics D008958:Models, Molecular D009154:Mutation D011392:Proline 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 D011994:Recombinant Proteins D013329:Structure-Activity Relationship D013379:Substrate Specificity D013816:Thermodynamics D014305:Triose-Phosphate Isomerase D014346:Trypanosoma brucei brucei D014349:Trypanosoma cruzi
differential scanning calorimetry contact maps activation energy TIM barrels beta-loop-alpha motifs beta-turns parasites protein engineering thermal unfolding