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Centro de Investigaciones en Química Biológica de Córdoba—CIQUIBIC, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Pabellón Argentina, Ciudad Universitaria, 5000 Córdoba, Argentina
Reprint requests to: Gerardo D. Fidelio, Centro de Investigaciones en Química Biológica de Córdoba—CIQUIBIC, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Pabellón Argentina, primer piso, Ciudad Universitaria, 5000 Córdoba, Argentina; e-mail: gfidelio{at}dqb.fcq.unc.edu.ar; fax: 54-351-433 4074.
The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted 
-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA1,8-ANS, BSA2,6-ANS, BSAfree, BSAbis-ANS, and BSAunfolded among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system.
Keywords: Protein stability; ligand binding; dynamic landscape; calorimetry; H/D exchange
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