Perturbing the unfolding of CRABP I using polyethylene glycol: An experimental and theoretical study

Proteins significantly affected by crowding nature of macromolecules. Thus, it is pertinent to investigate the role of crowding environment on the denaturation and renaturation kinetics of protein. Different molecular weights of Polyethylene glycol (PEG) have been considered as a molecular crowders and CRABP I (cellular retinoic acid binding protein I),as a model protein in this study. The secondary structure analysis was performed by circular dichroism (CD) spectroscopy and the unfolding kinetics using intrinsic fluorescence of CRABP I at 37o C to mimic the in vivo condition. Both PEG 2000 and PEG 4000 act as stabilizers by hamstring the unfolding kinetic rates. The unfolding kinetic slopes were different for both PEG, which indicating PEG favour compact conformations of protein as a function of concentration and molecular weight. The molecular dynamics(MD) studies revealed that PEG 2000 molecules assembled together during the 500 ns simulation, which is increasing the stability and percentage of s-sheet of the protein. The experimental findings were well supported by the theoretical results.