Monitoring mobility in the early steps of unfolding:: The case of oxidized cytochrome b5 in the presence of 2 M guanidinium chloride

被引:17
作者
Arnesano, F
Banci, L
Bertini, I
Koulougliotis, D
Monti, A
机构
[1] Univ Florence, CERM, Sesto Fiorentino, Italy
[2] Univ Florence, Dept Chem, Sesto Fiorentino, Italy
关键词
D O I
10.1021/bi992756k
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A Model-Free analysis of the N-15 relaxation properties of oxidized cytochrome bs, a heme-containing electron-transfer protein, has been performed in 2 M guanidinium chloride (GdmCl), i.e., just before the heme is released by the action of denaturant. This analysis provides information on the mobility in the nano- to picosecond time range. A parallel study on the motions in the milli- to microsecond time scale has also been performed by analyzing rotating-frame N-15 relaxation rates. The protein contains a 60:40 ratio of two conformers (A and B) differing for the rotation of the heme group around the alpha-gamma meso axis. The effect of denaturant has been followed for both species, and the mobility properties have been compared with the analogous information in the absence of denaturant. To complete the picture, we also performed N-15 relaxation measurements and the Model-Free analysis of the native B form, whereas data on the A form [Dangi, B., Sarma, S., Yan, C., Banville, D. L., Guiles, R. D. (1998) J. Phys. Chem. B 102, 8201-8208], as well as rotating-frame measurements for both native forms [Banci, L., Bertini, I., Cavazza, C., Felli, I. C., Koulougliotis, D. (1998) Biochemistry 37, 12320-12330; Arnesano, F., Banci, L., Bertini, I., Felli, I. C., Koulougliotis, D. (1999) Eur. J. Biochem, 260, 347-354], are already available in the literature. It is found that GdmCl tends to increase the internal mobility, although some residues are rigidified on both time scales. In the milli- to microsecond time scale, the tendency to increased mobility is reflected in a decrease in the tau(ex) values rather than in the number of residues experiencing conformational equilibria. In the nano- to picosecond time scale, the tendency to increased mobility is indicated by an overall decrease in the S-2 values. Color pictures are reported to visually show these effects. On the fast time scale, the B form is more mobile than the A form, reflecting the different stability with respect to unfolding. The increase in mobility upon addition of denaturant largely occurs around the heme pocket, which facilitates the release of the heme. The relevance of the internal motions with respect to the early steps of the unfolding process is also analyzed and discussed.
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页码:7117 / 7130
页数:14
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