Study of refolding of a denatured protein and microenvironment probed through FRET to a twisted intramolecular charge transfer fluorescent biosensor molecule

Abstract

In the present study, Fӧrster Resonance Energy Transfer (FRET) from Tryptophan residues (Trp) of bovine serum albumin (BSA) to a nongenotoxic and nontoxic molecule, trans-2-[4-(dimethylamino)styryl]benzothiazole (DMASBT) resulting in fluorescence from the twisted intramolecular charge transfer (TICT) state of the latter has been explored to probe the gemini surfactant induced unfolding and β-cyclodextrin (β-CD) induced refolding of the protein. DMASBT as a potential acceptor in FRET process with its polarity-sensitive TICT fluorescence properties is found to be an efficient biosensor molecule to mark the changes in microenvironment as well as conformations of the protein. λmax of TICT fluorescence band of DMASBT in native protein at 486 nm is red-shifted to 503 nm in unfolded protein which is blue-shifted to 487 nm upon refolding of the protein. Process of BSA fluorescence quenching through FRET to DMASBT has been utilized to compare between the hydrophobic area and fractional accessibility to quencher in native and refolded protein. Fluorescence quenching data have also been used to explain the binding affinity of DMASBT with native, refolded and unfolded BSA. Study reveals the roles of spacer group of gemini surfactants, 12-4-12,2Br− and 12-8-12,2Br− on refolding and unfolding of BSA. A gemini surfactant with a longer spacer is found to be easily stripped off by β-CD resulting in better refolding of protein gaining a larger hydrophobic area. Results obtained corroborate well with the findings from other experimental measurements on circular dichroism, dynamic light scattering and Hg2+ ion-induced fluorescence quenching. An easy and efficient method to study the microenvironment, and unfolding and refolding of a protein is presented here.