Conformational stability of cytochrome b5, enhanced green fluorescent protein, and their fusion protein Hmwb5-EGFP.
The conformational stabilities of chimeric protein Hmwb(5)-EGFP and its constituents (cytochrome b(5) and enhanced green fluorescent protein) in guanidine hydrochloride solutions are reported in this paper. Intensity of fluorescence of tryptophan residues, intensity of EGFP fluorescence in the visible region, absorbance of cytochrome b(5) heme and EGFP fluorophore, and fluorescence anisotropy were used to follow the unfolding process. Thermodynamic parameters of protein unfolding were obtained using different approaches. The data were analyzed using a two-stage model and a linear extrapolation method. Unfolding of protein molecules was additionally monitored by measuring Stern-Volmer constants for tryptophan fluorescence quenching by acrylamide, cesium, and iodide. The accessibility of tryptophan residues of both components in the fusion molecule is lower than in the separate molecules. The thermodynamic stability of the protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference in free energy of unfolding being more considerable for cytochrome b(5) (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 fusion can greatly affect total structural stability, and in the case of cytochrome b(5) and EGFP it results in decrease in free energy of transition from native to denatured unfolded form and consequently to decrease in thermodynamic stability of protein globules compared to the separate proteins.
Yantsevich
A V
AV
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
Gilep
A A
AA
Usanov
S A
SA
D000818:Animals; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015786:Cytochromes b5