Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase.
The conformational stability of the homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase (FabG) was determined by guanidinium chloride-induced isothermal and thermal denaturation. The reversible unfolding transitions were monitored by intrinsic fluorescence, circular dichroism (CD) spectroscopy and by measuring the enzyme activity of FabG. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with unfolding of the protein. The data confirm the simple A(4) <--> 4A model of unfolding, based on the corroboration of CD data by fluorescence transition and similar Delta G estimation for denaturation curves obtained at four different concentration of the FabG. Denaturation is well described by the linear extrapolation model for denaturant-protein interactions. In addition, the conformational stability (Delta G(s)) as well as the Delta C(p) for the protein unfolding is quite high, 22.68 kcal/mole and 5.83 kcal/(mole K), respectively, which may be a reflection of the relatively large size of the tetrameric molecule (Mr 120, 000) and a large buried hydrophobic core in the folded protein. This study provides a prototype for determining conformational stability of other members of the short-chain alcohol dehydrogenase/reductase superfamily of proteins to which PfFabG belongs.
Karmodiya
Krishanpal
K
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Sajad
Syed
S
Sinha
Sharmistha
S
Maity
Koustav
K
Suguna
Kaza
K
Surolia
Namita
N
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000502:physiology*; D000818:Animals; D004795:Enzyme Stability / Q000502:physiology; D010963:Plasmodium falciparum / Q000201:enzymology*; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D013816:Thermodynamics
D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase