Sucrose-Induced Stabilization of Domain-II and Overall Human Serum Albumin against Chemical and Thermal Denaturation

Abstract

In this contribution, we have compared the stabilizing effect of sucrose on overall as well as on domain-II of human serum albumin (HSA) against unfolding by different denaturating agents. HSA was denatured thermally by raising the temperature and also chemically by guanidine hydrochloride (GnHCl) and urea. Circular dichroism spectroscopy was used to monitor the change in the overall structure of HSA, whereas tryptophan fluorescence has been used to investigate the local structural alteration within the domain-II of HSA. The degree of folding using different combinations of GnHCl–sucrose, urea–sucrose, and temperature-sucrose was investigated to calculate the relative stability of the native, intermediate, and denatured states of HSA with increasing sucrose concentration. In the presence of sucrose, the intermediate state, formed during GnHCl-induced denaturation, populated at higher GnHCl concentration (1.5 M for overall denaturation and 2.5 M for domain-II) compared to that in the absence of sucrose (1 M for overall denaturation and 2 M for domain-II). A similar effect has also been observed for urea. These signify that sucrose stabilizes the native state of the protein. Extent of thermal unfolding is also found to be minimized in the presence of sucrose. In a nut shell, sucrose stabilizes different parts of HSA differently and also the net stabilizing effect toward different denaturation profiles is different.

Shape-Dependent Macromolecular Crowding on the Thermodynamics and Microsecond Conformational Dynamics of Protein Unfolding Revealed at the Single-Molecule Level

“…The autocorrelation curve for free NPCE has been fitted with eq satisfactorily, but to properly fit the autocorrelation curve for NPCE tagged to HSA, eq is required. The additional relaxation component arises from the microsecond conformational fluctuation dynamics of domain III of HSA around NPCE. ,,, Furthermore, we have recorded the autocorrelation curves of NPCE-tagged HSA between 15 and 70 °C in the absence and presence of various crowders. From the fluorescence intensity autocorrelation curve (Figure a), the hydrodynamic radius ( r H ) and conformational fluctuation time ( τ R ) are determined (Figure b,c and Table S4).…”

“…69 Earlier, we proved that the additional exponential time component of 8 μs in the fluorescence intensity autocorrelation of NPCE-tagged HSA arises from the conformational fluctuation dynamics of the side chain of domain III near NPCE. 34,35,58,59 Because of these dynamics, the surrounding electron-rich amino acid residues around NPCE come close to and far from the dye (NPCE) and modulate its fluorescence intensity. Such microsecond dynamics are a measure of the flexibility of the protein site and are directly correlated to its enzymatic activity.…”

“…We synthesized NPCE as described previously. ,,, The details can be found in section S1 of the Supporting Information.…”

“…56,57 It consists of three domains, and each of them behaves differently. 58,59 We have site-selectively tagged domain III of HSA at the Tyr-411 residue, which controls the esterase activity, with a coumarin dye (NPCE) 57,59−61 We have employed fluorescence correlation spectroscopic (FCS) experiments to decipher our proposition.…”

“…Dextran, a rod-shaped macromolecule, is made of glucose, , ficoll is a spherical macromolecule having multiple numbers of sucrose within it, ,, and PEG is a mesh-like polymer of monomeric ethylene glycol . The hydrodynamic radii of dextran-40, ficoll-70, and PEG-35 are 48, 51, and 55 Å, respectively, and roughly all three crowders used in the study can be represented as an equivalent sphere of hydrodynamic radius ∼50 Å, − at least when the crowder concentration is not too high (below the critical concentration). , HSA, with a hydrodynamic radius of around 40 Å, is the most abundant and most studied serum protein. , It consists of three domains, and each of them behaves differently. , We have site-selectively tagged domain III of HSA at the Tyr-411 residue, which controls the esterase activity, with a coumarin dye (NPCE) ,− We have employed fluorescence correlation spectroscopic (FCS) experiments to decipher our proposition.…”