Unfolding profiles of TvTIM1 and I45G mutant monitored by Intrinsic (black squares) and ANS (red circles) fluorescence

<p>. The conditions are as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141747#pone.0141747.g004" target="_blank">Fig 4</a>. The protein concentration was 50 μg ml^-1. Data are normalized for ease of comparison.</p

Ball and socket interaction between monomers at TvTIM1.

<p><b>(A)</b> Crystal structure of TvTIM1 showing the ball and socket interplay. Hydrophobic TvTIM1 residues (ball-stick representation) form a socket that interacts with residue Ile45 of the neighboring subunit (space-filling representation). The methyl group of I45 is colored in black. <b>(B)</b> Sequence logo showing the structural alignment of the ball-socket amino acids at the ball and socket interplay Residue I45 or V45 functions as the ball and a hydrophobic cavity formed by α-helices 2 and 3 that assemble as the socket.</p

Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from <i>Trichomonas vaginalis</i>

<div><p>The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Ų. TIMs from <i>Trichomonas vaginalis</i> (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.</p></div

Substrate dependent dimerization of monomeric TvTIMs.

<p>(A) Trace of absorbance at 280 nm of TvTIM1 during Sedimentation Velocity experiment at the upper panel followed by the residuals bitmaps. Symbols correspond to experimental data and lines are the results fitted to the Lamm equation using Sedfit [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141747#pone.0141747.ref028" target="_blank">28</a>]. The lower panel shows continuous (c(s)) distribution of wild-type TvTIM1 (black curve) and monomeric mutants I45A (red) and I45G (blue). The left dashed line indicates monomer position whereas the right one indicates dimer. (B) Oligomeric states of I45A mutant in the presence of increasing concentration of PGH. Continuous (c(s)) distribution of I45A mutants in 20 mM Tris-HCl pH 8.0 plus 50 mM NaCl buffer. The distributions of protein without substrate are shown in black lines; the ones with 20 μM of PGH are shown in red lines, with 250 μM of PGH in blue lines, with 600 μM of PGH in pink lines, with 1000 μM of PGH in green lines and with 2000 μM of PGH in dark blue lines.</p

<i>In vivo</i> characterization of TIMs complemented strains on glycerol minimal media in the presence of IPTG.

<p><b>(A)</b> Complementation of point mutants into an <i>E</i>. <i>coli</i> DE3 ΔTIM strain. Transformed <i>E</i>. <i>coli</i> were grown in plates with glycerol as a carbon source in M63 minimal media and 0.1mM of IPTG. <i>E</i>. <i>coli</i> transformed with plasmids containing wild-type TvTIM1 are able to complement. Mutants I45A, I45V, I45F and I45L complement with similar efficiency as wild type, whereas mutants I45Y contained less colonies, and no colonies appear after 48 hrs for I45G and I445W. <b>(B)</b> Growth kinetics of <i>E</i>. <i>coli</i> complemented strains in liquid minimal media. Growth rates of cultures grown in minimal medium. Bacteria transformed with plasmids containing wild-type TvTIM1 and mutants I45L, I45V, complement with similar efficiency, whereas, mutants I45A, I45F, I45Y and I45G present slower growth rates and no growth was observed with the tryptophan mutant and the empty vector.</p

The identity of residue 45 determines the dimer-monomer equilibrium of TvTIM1.

<p><b>(A)</b> Gel filtration elution profiles of wild-type and residue 45 point mutants. Mutants I45G, I45V, I45F and I45Y present as a monomer, whereas I45V and I45L as dimer. Mutant I45G and I45A present a small peak (6 and 3% of the total protein respectively) at the retention time of the dimer indicating that both mutants at a concentration of 260 μM exist in dimer-monomer equilibrium. <b>(B)</b> CD spectra of wild type and I45 mutants. The spectra of I45L and I45V superimpose with the wild-type spectra, whereas monomeric constructs present a decrease on ellipticity. <b>(C)</b> Partial proteolysis of TvTIM1 mutants. SDS-PAGE showing the digestion patterns of digested proteins after a partial proteolysis experiment (5 and 60 minutes). At 5 minutes almost the totality of the monomeric proteins I45G, I45V, I45F and I45Y are digested by trypsin, and after 60 minutes the digestion is complete. In contrast a strong protein band is observed for wild-type and of I45L and I45V mutants indicating that dimeric enzymes present an increased resistance to proteolysis.</p

Thermodynamic Parameters for TvTIM1 and I45X mutants.

<p>^a Measurements were made at 25°C and pH 7.4. Global analysis was performed with the non-linear, least-squares fitting program Origin, version 8.0. Standard deviations are indicated in parentheses.</p><p>^b calculated according to the relationship <math><mrow><mo>Δ</mo><msubsup><mi>G</mi><mrow><mi>I</mi><mi>D</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup><mo>=</mo><mo>Δ</mo><msubsup><mi>G</mi><mrow><mi>N</mi><mi>D</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup><mo>−</mo><mo>Δ</mo><msubsup><mi>G</mi><mrow><mi>N</mi><mi>I</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup></mrow></math></p><p>^c estimated using equation <math><mrow><mo>Δ</mo><msubsup><mi>G</mi><mrow><msub><mi>N</mi><mn>2</mn></msub><mn>2</mn><mi>D</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup><mo>=</mo><mo>Δ</mo><msubsup><mi>G</mi><mrow><msub><mi>N</mi><mn>2</mn></msub><mn>2</mn><mi>M</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup><mo>+</mo><mn>2</mn><mo>Δ</mo><msubsup><mi>G</mi><mrow><mi>M</mi><mi>I</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup><mo>+</mo><mn>2</mn><mo>Δ</mo><msubsup><mi>G</mi><mrow><mi>I</mi><mi>D</mi></mrow><mrow><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></mrow></msubsup></mrow></math></p><p>Thermodynamic Parameters for TvTIM1 and I45X mutants.</p

Cavities at the ball and socket interaction by mutations of residue 45.

<p>Cavities at the ball and socket interaction by mutations of residue 45.</p

Monomeric TvTIMs are partially resistant to proteolysis upon 2PGA incubation.

<p>Time course experiment showing the partial proteolysis patterns of TvTIM mutants in the absence and in the presence of 2PGA. Dimeric TvTIMs are resistant to trypsinolysis with or without substrate analog (A and D). However monomeric TvTIMs are susceptible to trypinolysis in the absence of 2PGA but a small population of monomeric TvTIMs become resistant to after incubation with 2PGA (B,C,E,F). The extend of proteolysis is derived from the comparison to the control protein without added protease (lane 1).</p

AUC data analysis for I45A mutant in 20 mM Tris-HCl.

<p>pH 8.0 and 50 mM NaCl with different concentrations of PGH</p><p>AUC data analysis for I45A mutant in 20 mM Tris-HCl.</p