Limits...
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.

Show MeSH

Related in: MedlinePlus

Disulfide Cross-linker induced dimerization of I45G mutant.(A) Localization of the engineered I45G-Q52C double mutant. The upper panel represents the monomeric I45G-Q52C double mutatn that may assemble as a dimer by effect of a cross-linking reaction (lower panel) (B) Gel filtration elution profiles and SDS-PAGE analysis for wild-type and I45G-Q52C double mutant before and after cross-linking. Approximately two thirds of the I45G-Q52C double mutant cross-linked in to a dimer as assessed by SDS-PAGE (lane 3 bottom panel). The differences between the percentage of assembled dimer by SDS-PAGE and gel filtration may due to a differential in the exposure of aromatic residues between the monomer and dimer (C) Gel filtration elution profiles and SDS-PAGE for wild-type and purified cross-linked I45G-Q52C double mutant. The gel filtration step efficiently separates the cross-linked I45G-Q52C double mutant from the unreacted protein (lane 1 bottom panel). The elution profiles have been normalized to ease comparison.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664265&req=5

pone.0141747.g010: Disulfide Cross-linker induced dimerization of I45G mutant.(A) Localization of the engineered I45G-Q52C double mutant. The upper panel represents the monomeric I45G-Q52C double mutatn that may assemble as a dimer by effect of a cross-linking reaction (lower panel) (B) Gel filtration elution profiles and SDS-PAGE analysis for wild-type and I45G-Q52C double mutant before and after cross-linking. Approximately two thirds of the I45G-Q52C double mutant cross-linked in to a dimer as assessed by SDS-PAGE (lane 3 bottom panel). The differences between the percentage of assembled dimer by SDS-PAGE and gel filtration may due to a differential in the exposure of aromatic residues between the monomer and dimer (C) Gel filtration elution profiles and SDS-PAGE for wild-type and purified cross-linked I45G-Q52C double mutant. The gel filtration step efficiently separates the cross-linked I45G-Q52C double mutant from the unreacted protein (lane 1 bottom panel). The elution profiles have been normalized to ease comparison.

Mentions: We reasoned that the reduced activity of the monomeric mutant is a consequence of deficiencies in dimer formation and that substrate binding of other forces may be able to restore the dimer. For instance, the crowding effect increases the formation of dimeric TIMs from from unfolded monomers [35]. Chemically cross-linking has been used to stabilize monomeric variants of Cu/Zn superoxide dismutase [36]. Mutant I45G reduces its catalytic efficiency 142-fold in comparison to wild-type TvTIM1 (Table 1) and we reason that a covalent cross-linking of the monomeric I45G mutant may increase its activity. For site-directed mutagenesis we selected residues Q52 and K53 since their C-β between monomers are 11 and 13 Å separated. We assayed dithio-bismaleimidoethane (DTME) and 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB) as cross-linkers with space arms of 13.3 and 10.2 Å respectively (Fig 10A).


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)

Disulfide Cross-linker induced dimerization of I45G mutant.(A) Localization of the engineered I45G-Q52C double mutant. The upper panel represents the monomeric I45G-Q52C double mutatn that may assemble as a dimer by effect of a cross-linking reaction (lower panel) (B) Gel filtration elution profiles and SDS-PAGE analysis for wild-type and I45G-Q52C double mutant before and after cross-linking. Approximately two thirds of the I45G-Q52C double mutant cross-linked in to a dimer as assessed by SDS-PAGE (lane 3 bottom panel). The differences between the percentage of assembled dimer by SDS-PAGE and gel filtration may due to a differential in the exposure of aromatic residues between the monomer and dimer (C) Gel filtration elution profiles and SDS-PAGE for wild-type and purified cross-linked I45G-Q52C double mutant. The gel filtration step efficiently separates the cross-linked I45G-Q52C double mutant from the unreacted protein (lane 1 bottom panel). The elution profiles have been normalized to ease comparison.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141747.g010: Disulfide Cross-linker induced dimerization of I45G mutant.(A) Localization of the engineered I45G-Q52C double mutant. The upper panel represents the monomeric I45G-Q52C double mutatn that may assemble as a dimer by effect of a cross-linking reaction (lower panel) (B) Gel filtration elution profiles and SDS-PAGE analysis for wild-type and I45G-Q52C double mutant before and after cross-linking. Approximately two thirds of the I45G-Q52C double mutant cross-linked in to a dimer as assessed by SDS-PAGE (lane 3 bottom panel). The differences between the percentage of assembled dimer by SDS-PAGE and gel filtration may due to a differential in the exposure of aromatic residues between the monomer and dimer (C) Gel filtration elution profiles and SDS-PAGE for wild-type and purified cross-linked I45G-Q52C double mutant. The gel filtration step efficiently separates the cross-linked I45G-Q52C double mutant from the unreacted protein (lane 1 bottom panel). The elution profiles have been normalized to ease comparison.
Mentions: We reasoned that the reduced activity of the monomeric mutant is a consequence of deficiencies in dimer formation and that substrate binding of other forces may be able to restore the dimer. For instance, the crowding effect increases the formation of dimeric TIMs from from unfolded monomers [35]. Chemically cross-linking has been used to stabilize monomeric variants of Cu/Zn superoxide dismutase [36]. Mutant I45G reduces its catalytic efficiency 142-fold in comparison to wild-type TvTIM1 (Table 1) and we reason that a covalent cross-linking of the monomeric I45G mutant may increase its activity. For site-directed mutagenesis we selected residues Q52 and K53 since their C-β between monomers are 11 and 13 Å separated. We assayed dithio-bismaleimidoethane (DTME) and 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB) as cross-linkers with space arms of 13.3 and 10.2 Å respectively (Fig 10A).

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