CONFORMATIONAL STABILITY OF CYTOCHROME <i>b</i><sub><i>5 </i></sub>AND ENHANCED GREEN FLUORESCENT PROTEIN AND THEIR FUSION PROTEIN Hmwb<sub>5</sub>-EGFP
In the present article conformational stabilities of chimeric protein Hwmb5-EGFP and its constituents cytochrome b5 and EGFP in guanidine hydrochloride solution are reported. The intensity of fluorescence of tryptophan, intensity of EGFP fluorescence in the visible region, absorption of cytochrome b5 heme and EGFP fluorophore, and fluorescence anisotropy were used to monitor unfolding. Thermodynamic parameters of protein unfolding were determined using different approaches. For data analysis a two stage model and linear extrapolation method was used. Unfolding of protein molecules in addition was monitored by measuring Stern-Volmer constant for tryptophan fluorescence quenching by acrylamide and cesium and iodine ions. It was found that accessibility of tryptophan moieties of both components in the structure of the fusion molecule is lower than in the initial molecules. It is shown that thermodynamic stability of protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference of free energy of unfolding being more considerable for cytochrome b5 (29 ± 4 and 13 ± 2 kJ/mol) than for EGFP (26 ± 0.9 and 20 ± 2.7 kJ/mol). The data indicate that artificial protein conjugation may greatly affect structure stability.