Region-Specific Double Denaturation of Human Serum Albumin: Combined Effects of Temperature and GnHCl on Structural and Dynamical Responses.

In this work, we have investigated the effects of denaturing agents, guanidine hydrochloride (GnHCl) and temperature, on the overall structure, domain-I, and domain-III of human serum albumin (HSA) using circular dichroism (CD) spectroscopy and steady-state, time-resolved fluorescence spectroscopy. We have tagged Cys-34 of HSA, located at domain-I, using -(7-dimethylamino-4-methylcoumarin-3-yl)iodoacetamide and Tyr-411 of HSA, located at domain-III, using -nitrophenyl coumarin ester, for this purpose. The CD spectroscopy studies reveal the overall denaturation of the protein. The denaturation follows the expected direction in which the protein is denatured with an increase in the concentration of GnHCl or temperature. The α-helicity of the native state of HSA was found to be 64.2%, and the minimum value of α-helicity was found to be 14.8% in the presence of 6 M GnHCl at room temperature. Steady-state emission studies were carried out on domain-I and domain-III of the protein using site-specific fluorescent tags. The degree of folding of the two domains at different combinations of temperature and GnHCl concentration was calculated and was found to follow a slightly different course of denaturation. Solvation dynamics was found to be quite different for these two domains. The domain-I of HSA has a maximum solvation time of 0.39 ns, and the solvation time tends to decrease with the action of either temperature or GnHCl. On the other hand, the domain-III of HSA showed a much higher solvation time (1.42 ns) and does not show any regular change at higher temperatures or in the presence of GnHCl. This difference could be attributed to the different microenvironment inside the protein cores of the two domains.