Journal
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
Volume 85, Issue 4, Pages 571-579Publisher
WILEY
DOI: 10.1002/prot.25231
Keywords
beta-turns; beta-loop-alpha motifs; contact maps; differential scanning calorimetry; activation energy; protein engineering; TIM barrels; thermal unfolding; parasites
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Funding
- SNI
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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 beta-turn occurrence, we engineered two chimerical enzymes where their super secondary beta-loop-alpha motifs 2 ((beta alpha)(2)) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (beta alpha)(2) motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (beta alpha)(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 (beta alpha)(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 alpha-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (E-A). A systematic analysis of DSC data showed a large decrease on the E-A of TcTIM (Delta E-A 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. (C) 2016 Wiley Periodicals, Inc.
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