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Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (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.

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Substrate dependent dimerization of monomeric TvTIMs.(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 [28]. 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.
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pone.0141747.g009: Substrate dependent dimerization of monomeric TvTIMs.(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 [28]. 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.

Mentions: In order to understand if the proteolysis resistance of the monomeric mutants is due to the formation of a dimer, we performed sedimentation velocity experiments using wild-type TvTIM1 and I45A and I45G mutants over a concentration range from 7 to 35 μM show that the TvTIM1 is a dimer in solution, whereas I45A and I45G are monomers (Fig 9A).


Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

Substrate dependent dimerization of monomeric TvTIMs.(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 [28]. 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664265&req=5

pone.0141747.g009: Substrate dependent dimerization of monomeric TvTIMs.(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 [28]. 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.
Mentions: In order to understand if the proteolysis resistance of the monomeric mutants is due to the formation of a dimer, we performed sedimentation velocity experiments using wild-type TvTIM1 and I45A and I45G mutants over a concentration range from 7 to 35 μM show that the TvTIM1 is a dimer in solution, whereas I45A and I45G are monomers (Fig 9A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (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.

Show MeSH
Related in: MedlinePlus