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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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The identity of residue 45 determines the dimer-monomer equilibrium of TvTIM1.(A) 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) 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. (C) 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.
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pone.0141747.g002: The identity of residue 45 determines the dimer-monomer equilibrium of TvTIM1.(A) 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) 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. (C) 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.

Mentions: We measured the oligomeric state of the ball-socket mutants using gel filtration at a concentration of 7 mg/ml (~260 μM) in Tris pH 7.4, 100 mM NaCl (Fig 2A).


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)

The identity of residue 45 determines the dimer-monomer equilibrium of TvTIM1.(A) 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) 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. (C) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141747.g002: The identity of residue 45 determines the dimer-monomer equilibrium of TvTIM1.(A) 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) 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. (C) 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.
Mentions: We measured the oligomeric state of the ball-socket mutants using gel filtration at a concentration of 7 mg/ml (~260 μM) in Tris pH 7.4, 100 mM NaCl (Fig 2A).

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