Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: Solvation dynamics study

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the solvation dynamics of a probe covalently attached to a protein (human serum albumin (HSA)) has been studied using femtosecond up-conversion. For this study, a solvation probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) has been covalently attached to the lone cysteine group (cys-34) of the protein HSA. Addition of 1.5 M RTIL or 6 M GdnHCl causes a red shift of the emission maxima of CPM bound to HSA by 3 nm and 12 nm, respectively. The average solvation time ⟨τ_s⟩ decreases from 650 ps (in native HSA) to 260 ps (∼2.5 times) in the presence of 1.5 M RTIL and to 60 ps (∼11 times) in the presence of 6 M GdnHCl. This is ascribed to unfolding of the protein by RTIL or GdnHCl and therefore making the probe CPM more exposed. When 1.5 M RTIL is added to the protein denatured by 6 M GdnHCl in advance, a further ∼5 nm red shift along with further ∼2 fold faster solvent relaxation (⟨τ⟩ ∼30 ps) is observed. Our previous fluorescence correlation spectroscopy study [D. K. Sasmal, T. Mondal, S. Sen Mojumdar, A. Choudhury, R. Banerjee, and K. Bhattacharyya, J. Phys. Chem. B 115, 13075 (2011) https://doi.org/10.1021/jp207829y] suggests that addition of RTIL to the protein denatured by 6 M GdnHCl causes a reduction in hydrodynamic radius (r_h). It is demonstrated that in the presence of RTIL and GdnHCl, though the protein is structurally more compact, the local environment of CPM is very different from that in the native state.