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Half-Barrels Derived from a (β/α)8 Barrel β-Glycosidase Undergo an Activation Process.

Beton D, Marana SR - PLoS ONE (2015)

Bottom Line: The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively.Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles.These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
The evolution of (β/α)8 barrel proteins is currently thought to have involved the fusion of two (β/α)4 half-barrels, thereby conferring stability on the protein structure. After the formation of a whole (β/α)8 barrel, this structure could evolve and diverge to form fully active enzymes. Interestingly, we show here that isolated (β/α)4 half-barrels derived from the N- and C-terminal domains of the β-glucosidase Sfβgly (Sfβgly-N: residues 1 to 265; Sfβgly-C: residues 266 to 509) undergo an activation process, which renders them catalytically active. The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively. Moreover, the Sfβgly-N and Sfβgly-C activation processes were simultaneous with modifications in their initial structure, which reduced the exposure of their tryptophan residues. Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles. These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

No MeSH data available.


Related in: MedlinePlus

Expression and purification of Sfβgly-N and Sfβgly-C.Proteins from the soluble (lane S) and insoluble (lane P) fractions of bacteria (ArticExpress (DE3)) induced to produce Sfβgly-N (A) and Sfβgly-C (B) were analyzed by SDS–PAGE (12% polyacrylamide; Comassie Blue R250 staining), transferred to a nitrocellulose membrane and immuno-detected using anti-His6 antibody (lanes 1 and 2, respectively). Sfβgly-N and Sfβgly-C were purified from the respective soluble fractions using Ni-NTA agarose and analyzed by SDS–PAGE (lane 3; arrow; Comassie Blue R250 staining).
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pone.0139673.g001: Expression and purification of Sfβgly-N and Sfβgly-C.Proteins from the soluble (lane S) and insoluble (lane P) fractions of bacteria (ArticExpress (DE3)) induced to produce Sfβgly-N (A) and Sfβgly-C (B) were analyzed by SDS–PAGE (12% polyacrylamide; Comassie Blue R250 staining), transferred to a nitrocellulose membrane and immuno-detected using anti-His6 antibody (lanes 1 and 2, respectively). Sfβgly-N and Sfβgly-C were purified from the respective soluble fractions using Ni-NTA agarose and analyzed by SDS–PAGE (lane 3; arrow; Comassie Blue R250 staining).

Mentions: The cDNA coding the N- and C-terminal halves (residues 1 to 265 and 266 to 509) of the GH1 β-glucosidase Sfβgly [9] (AF052729) was amplified by PCR and cloned into the expression vector pAE. Considering that Sfβgly folds as a (β/α)8 barrel, these halves are hereafter termed Sfβgly-N and Sfβgly-C, which are predicted to correspond to (β/α)4 half-barrels. Thus, each of these half-barrels, as well as native Sfβgly, were produced as recombinant proteins in ArticExpress(DE3) bacteria at 12°C for 24 h. Next, they were successfully purified using Ni-NTA agarose resin. The homogeneity and identity of Sfβgly-N and Sfβgly-C were confirmed by SDS–PAGE and western blotting using anti-His6 antibody (Fig 1). Approximately 200 μg of both soluble Sfβgly-N and Sfβgly-C was obtained from 3 L induced bacterial media. However, the yields were variable, and freshly transformed bacteria had to be used for each induction. Notably, Sfβgly-N and Sfβgly-C were also expressed in BL21-Gold (DE3) bacteria at 20, 25 and 37°C, but no soluble recombinant protein was obtained from those induced bacteria.


Half-Barrels Derived from a (β/α)8 Barrel β-Glycosidase Undergo an Activation Process.

Beton D, Marana SR - PLoS ONE (2015)

Expression and purification of Sfβgly-N and Sfβgly-C.Proteins from the soluble (lane S) and insoluble (lane P) fractions of bacteria (ArticExpress (DE3)) induced to produce Sfβgly-N (A) and Sfβgly-C (B) were analyzed by SDS–PAGE (12% polyacrylamide; Comassie Blue R250 staining), transferred to a nitrocellulose membrane and immuno-detected using anti-His6 antibody (lanes 1 and 2, respectively). Sfβgly-N and Sfβgly-C were purified from the respective soluble fractions using Ni-NTA agarose and analyzed by SDS–PAGE (lane 3; arrow; Comassie Blue R250 staining).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591997&req=5

pone.0139673.g001: Expression and purification of Sfβgly-N and Sfβgly-C.Proteins from the soluble (lane S) and insoluble (lane P) fractions of bacteria (ArticExpress (DE3)) induced to produce Sfβgly-N (A) and Sfβgly-C (B) were analyzed by SDS–PAGE (12% polyacrylamide; Comassie Blue R250 staining), transferred to a nitrocellulose membrane and immuno-detected using anti-His6 antibody (lanes 1 and 2, respectively). Sfβgly-N and Sfβgly-C were purified from the respective soluble fractions using Ni-NTA agarose and analyzed by SDS–PAGE (lane 3; arrow; Comassie Blue R250 staining).
Mentions: The cDNA coding the N- and C-terminal halves (residues 1 to 265 and 266 to 509) of the GH1 β-glucosidase Sfβgly [9] (AF052729) was amplified by PCR and cloned into the expression vector pAE. Considering that Sfβgly folds as a (β/α)8 barrel, these halves are hereafter termed Sfβgly-N and Sfβgly-C, which are predicted to correspond to (β/α)4 half-barrels. Thus, each of these half-barrels, as well as native Sfβgly, were produced as recombinant proteins in ArticExpress(DE3) bacteria at 12°C for 24 h. Next, they were successfully purified using Ni-NTA agarose resin. The homogeneity and identity of Sfβgly-N and Sfβgly-C were confirmed by SDS–PAGE and western blotting using anti-His6 antibody (Fig 1). Approximately 200 μg of both soluble Sfβgly-N and Sfβgly-C was obtained from 3 L induced bacterial media. However, the yields were variable, and freshly transformed bacteria had to be used for each induction. Notably, Sfβgly-N and Sfβgly-C were also expressed in BL21-Gold (DE3) bacteria at 20, 25 and 37°C, but no soluble recombinant protein was obtained from those induced bacteria.

Bottom Line: The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively.Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles.These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
The evolution of (β/α)8 barrel proteins is currently thought to have involved the fusion of two (β/α)4 half-barrels, thereby conferring stability on the protein structure. After the formation of a whole (β/α)8 barrel, this structure could evolve and diverge to form fully active enzymes. Interestingly, we show here that isolated (β/α)4 half-barrels derived from the N- and C-terminal domains of the β-glucosidase Sfβgly (Sfβgly-N: residues 1 to 265; Sfβgly-C: residues 266 to 509) undergo an activation process, which renders them catalytically active. The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively. Moreover, the Sfβgly-N and Sfβgly-C activation processes were simultaneous with modifications in their initial structure, which reduced the exposure of their tryptophan residues. Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles. These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

No MeSH data available.


Related in: MedlinePlus