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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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Monomeric TvTIMs are partially resistant to proteolysis upon 2PGA incubation.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).
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pone.0141747.g008: Monomeric TvTIMs are partially resistant to proteolysis upon 2PGA incubation.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).

Mentions: As monomeric TvTIMs display high enzymatic activity and assemble as a dimer in the crystal enviroment, we wonder if the addition of a substrate analog would alter their oligomeric state in solution. A partial proteolysis experiment from 5 to 60 minutes was performed in the presence of 2 mM of the competitive inhibitor 2PGA (Fig 8).


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)

Monomeric TvTIMs are partially resistant to proteolysis upon 2PGA incubation.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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141747.g008: Monomeric TvTIMs are partially resistant to proteolysis upon 2PGA incubation.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).
Mentions: As monomeric TvTIMs display high enzymatic activity and assemble as a dimer in the crystal enviroment, we wonder if the addition of a substrate analog would alter their oligomeric state in solution. A partial proteolysis experiment from 5 to 60 minutes was performed in the presence of 2 mM of the competitive inhibitor 2PGA (Fig 8).

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