2,2,2-trifluoroethanol-induced molten globule state of concanavalin a and energetics of 8-anilinonaphthalene sulfonate binding: calorimetric and spectroscopic investigation.
The interaction of 2,2,2-trifluoroethanol (TFE) with concanavalin A has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy at pH 2.5 and 5.2. All of the calorimetric transitions at both the pH values were found to be irreversible. In the presence of 4 mol kg(-1) TFE at pH 2.5, concanavalin A is observed to be in a partially folded state with significant loss of native tertiary structure. The loss of specific side chain interactions in the transition from native to the TFE-induced partially folded state is demonstrated by the loss of cooperative thermal transition and reduction of the CD bands in the aromatic region. Acrylamide quenching, 8-anilinonaphthalene sulfonate (ANS) binding, and energy transfer also suggest that in the presence of 4 mol kg(-1) TFE at pH 2.5 concanavalin A is in a molten globule state. ITC has been used for the first time to characterize the energetics of ANS binding to the molten globule state. ITC results indicate that the binding of ANS to the molten globule state and acid-induced state at pH 2.5 displays heterogeneity with two classes of non-interacting binding sites. The results provide insights into the role of hydrophobic and electrostatic interactions in the binding of ANS to concanavalin A. The results also demonstrate that ITC can be used to characterize the partially folded states of the protein both qualitatively and quantitatively.
Banerjee
Tuhina
T
Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
Kishore
Nand
N
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D003208:Concanavalin A / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D014270:Trifluoroethanol / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D003208:Concanavalin A; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol