Elucidation of μs dynamics of domain-III of human serum albumin during the chemical and thermal unfolding: A fluorescence correlation spectroscopic investigation

- Denaturation of domain-III of HSA is studied by covalent marker for the first time.
- Global and domain-III denaturation were found to follow different pathways.
- Intermediate state/s during the denaturation is detected within the domain-III.
- Effects of GnHCl and heat on domain-III of HSA are found to be different.
- Conformational dynamics during the course of denaturation has been elucidated.

The local structural dynamics and denaturation profile of domain-III of HSA against guanidine hydrochloride (GnHCl) and temperature has been studied using a coumarin based solvatochromic fluorescent probe p-nitrophenyl coumarin ester (NPCE), covalently tagged to Tyr-411 residue. By the steady state, time-resolved and single molecular level fluorescence studies it has been established that the domain-III of HSA is very sensitive to GnHCl but somewhat resistant to temperature and the domain specific unfolding proceeds in an altered way as compared to the overall unfolding of HSA. While the overall denaturation of HSA is a two-state process for both GnHCl and heat, domain-III adopts two intermediate states for GnHCl induced denaturation and one intermediate state for temperature induced denaturation. Fluorescence correlation spectroscopic investigation divulges the conformational dynamics of domain-III of HSA in the native, intermediates and denatured state.

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