Effect of MMTS on WT TbTIM, WT TcTIM and on different site directed mutants of selected amino acids of regions 1 and 4 on the sequence of WT TbTIM.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: F28L, A100L, A115Q. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: A100L, A115Q, TbTIM: F28L, A100L and TbTIM: F28L, A115Q. Panel c) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: F28L, TbTIM: A100L and TbTIM: A115Q. Assays were performed independently three times.</p

pKa values of Cys 14/15 in WT TbTIM and WT TcTIM and some mutant enzymes.

<p>pKa values of Cys 14/15 in WT TbTIM and WT TcTIM and some mutant enzymes.</p

Effect of MMTS on WT TbTIM, WT TcTIM and on different site directed mutants of selected amino acids of regions 1 and 4 of the sequence of WT TbTIM onto WT TcTIM.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM and TcTIM: E26D, T27L, L28F, A30S, T32S, L100A, Q115A, TcTIM: E26D, T27L, L28F, A30S, L100A, Q115A and TcTIM: E26D, T27L, L28F, L100A, Q115A. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and TcTIM: E26D, T27L, L28F, A30S, T32S, L100A; TcTIM: E26D, T27L, L28F, A30S, L100A and TcTIM: E26D, T27L, L28F, L100A. Assays were performed independently three times.</p

Location of regions 1 and 4 in a ribbon diagram of the structure of WT TbTIM and aligned sequences of regions 1 and 4 of WT TbTIM and WT TcTIM.

<p>The ribbon diagram of the structure of 5TIM in the PDB database, corresponding to a dimer of WT TbTIM, is shown in red. Regions 1 and 4 are shown in blue and marine blue for monomers A and B, respectively. The n-terminus of each monomer and the position of amino acids 35, 92 and 119 are indicated by the corresponding text or number, sometimes using an arrow, on the diagram. The interface cysteines of both monomers are shown as yellow spheres. In the alignments, the differences in the amino acids are highlighted as conservative (similar size and polarity) in grey, semiconservative (similar polarity) in green, and without similarity in cyan. Secondary structure elements are shown below as dark green lines (loops), arrows (beta sheets) and barrels (alpha helixes).</p

Effect of MMTS on WT TbTIM, WT TcTIM and on different additive mutants of region 4.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM, R4M1 and TbTIM 1–3, 5–8; TcTIM 4. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and R4M2, R4M3, R4M4 and R4M5. Assays were performed independently three times.</p

Effect of DTDA on the activity of TIMs from <i>T. cruzi, T. brucei, L. mexicana</i> and <i>Homo sapiens</i>.

<p>The enzymes were incubated at a concentration of 5 µg/ml in media that contained 100 mM triethanolamine, 10 mM EDTA, 10% DMSO (v/v) and the indicated concentrations of DTDA. After two hours of incubation at 36°C, aliquots were withdrawn for assay of activity. 100% corresponds to the activity of TcTIM, TbTIM, LmTIM, and human TIM at time zero; these activities were 2950, 3125, 4560, and 4900 µmol/min/mg, respectively.</p

Reaction of DTDA with cysteine.

<p>The reaction of DTDA with the thiol group of Cys could be favored by the formation of an intramolecular hydrogen bond and the inductive effect of the amino group due to the higher electronegativity of the nitrogen atom with respect to the sulfur atom.</p

Structural alterations of the interfacial Cys15-loop3 regions of TcTIM induced by the soaking with DTDA.

<p>Panel A shows the superposition of loop3 of monomers A and B of the TcTIM crystal soaked with DTDA. It depicts the large scale difference in the conformations of loop3 of the “normal” monomer (B, in blue), and the “open” loop3 of monomer A (in green) in the span of residues 70–76. The hinge residues T70 and T76 are colored in orange. Panel B shows the interface region formed by Cys15 of monomer B and loop3 of the other monomer in native TcTIM. Panel C shows the same region in the DTDA treated enzyme. Please note that in native TcTIM, Cys15 is well defined, and that it establishes two H-bonds with Gly73 of the adjacent subunit (black dashed lines). These H-bonds do not exist in the DTDA treated enzyme. Electron density maps are included (2Fo-Fc, 1σ in blue and Fo-Fc, -3σ red), showing instability of Cys 15B.</p

Data Collection and Reduction Statistics for DTDA treated TcTIM.

<p>R = Σ|Fo-Fc|/ΣFo, summed over all hkl's</p

Effect of DTDA on <i>T.cruzi</i> epimastigotes.

<p>The parasites were cultured as described under <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000001#s2" target="_blank">Material and Methods</a> with the indicated concentrations of DTDA.</p