Probing deuterium isotope effect on structure and solvation dynamics of human serum albumin

Abstract

The deuterium isotopic effect on the structure and solvation dynamics of the protein, human serum albumin (HSA), has been studied by using circular dichroism (CD), femtosecond up-conversion, FRET, and single-molecule spectroscopy. The CD spectra suggest that D₂O affects the structure of HSA, leading to a 20% decrease in the helical structure. The FRET study indicates that the distance of C153 from the lone tryptophan residue of HSA is quite similar ( ≈21 Å) in H₂O and D₂O, and hence, the location of the probe in the protein remains the same in the two solvents. The single-molecule study suggests that coumarin 153 (C153) binds almost exclusively (>96%) to one site of HSA. Solvation dynamics of C153 in HSA is found to be markedly retarded in D₂O compared with H₂O. In H₂O, the solvation of C153 bound to HSA is found to be biexponential with one component of 7 ps (30%) and a long component of 350 ps (70%). In D₂O, we detected a short component of 4 ps (41%) and a long component of 950 ps (59%). Thus, the ultraslow component of the solvation dynamics of C153 bound to HSA in D₂O (950 ps) is 2.5-fold slower than that in H₂O (350 ps). The marked deuterium isotope effect has been ascribed to water molecules confined in the protein environment and to a lesser extent to the structural modification of protein by D₂O.