Methanol-induced unfolding and refolding of cytochrome b5 and its P40V mutant monitored by UV-visible, CD, and fluorescence spectra.
In order to illustrate the structural importance of proline-40 of cytochrome b5 (Cyt b5), the P40V mutant gene was constructed. Unfolding and refolding of Cyt b5 induced by methanol was investigated by means of the UV-visible spectrum, circular dichroism, and the fluorescence spectrum. Methanol denaturation of Cyt b5 is a cooperative process, that is, the heme group dissociates from the heme pocket accompanied by unfolding of the polypeptide chain both in the secondary and tertiary structures. Substitution of proline by valine reduces the stability of the mutant under methanol denaturation. The unfolding process is almost reversible by dilution. During refolding, the denatured polypeptide must be folded to a more ordered structure prior to the heme capture. Pro40 plays an important role in modulating the protein's stability. The role of tyrosine in the unfolding and refolding of Cyt b5 is evaluated for the first time. A mechanism of methanol denaturation is also proposed.
Wang
Z Q
ZQ
Chemistry Department, Fudan University, Shanghai, China.
Wang
Y H
YH
Qian
W
W
Wang
H H
HH
Chunyu
L J
LJ
Xie
Y
Y
Huang
Z X
ZX
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D015786:Cytochromes b5 / Q000737:chemistry*; D000432:Methanol / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D015786:Cytochromes b5; D000432:Methanol