Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation.
Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation.
复制标题
通过蛋氨酸 65 的邻硝基苄基修饰和随后的紫外光照射研究细胞色素 c 的折叠特性。
DOI：
10.1021/bi000305q
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
O. Yamauchi
中科院分区：
文献类型：
--
作者：
T. Okuno;S. Hirota;O. Yamauchi
用一种可光解的邻硝基苯并甲基65(NBz-Met65)研究了细胞色素c的蛋白质折叠特性。对于NBz-Met65cytc，与未标记的cytc相比，Soret的吸收带略有蓝移，与Met80硫键连接到血红素铁的695 nm吸收带消失，其共振拉曼光谱具有六配位低自旋物种的特征，所有特征都表明了非天然配体的配位，可能是组氨酸，而不是Met80对血红素铁的配位。修饰改变了Cytc的远紫外圆二色谱，使蛋白质解离所需的盐酸胍(GdnHCl)的过渡点浓度降低了0.9M，分别表现为结构的扰动和蛋白质稳定性的降低。在308 nm激光照射下，NBz-Met65细胞色素C的Soret吸收带略有红移，695 nm吸收峰出现，CD光谱向天然蛋白质方向移动，表明NBz-Met65重新转化为未标记的蛋氨酸，恢复了蛋氨酸的血红素配位和天然蛋白质结构。在2M GdnHCl存在下，308 nm脉冲辐照NBz-Met65Cytc引发的细胞色素c复性过程中，Soret吸光度的变化为快相，其速率常数为21000+/-4000 S(-)(1)。观察到的速率常数与色氨酸荧光研究[Shastry，M.C.R.S.和Roder，H.(1998)NAT]报道的一致。结构。比奥尔。5,385-392]。中间体衰变的速率常数为90+/-15，然后是另一相，速率常数为13+/-3 S(-)(1)，在没有GdnHCl的情况下看不到中间相。
The protein folding character of cyt c was studied with the use of a photocleavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 cyt c, the Soret absorption band slightly blue shifted compared with the unlabeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligation to the heme iron disappeared, and its resonance Raman spectrum was characteristic of a six-coordinate low-spin species, all characters demonstrating coordination of a non-native ligand, probably a histidine, instead of Met80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c was altered, and the transition midpoint concentration value of guanidine hydrochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the modification, which showed perturbation of the structure and decrease in protein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 nm absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordination and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorbance with a rate constant of 21 000 +/- 4000 s(-)(1) during refolding of cyt c initiated by irradiation of a 308 nm pulse on the NBz-Met65 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with that reported by the tryptophan fluorescence study [Shastry, M. C. R. S., and Roder, H. (1998) Nat. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate constant of 13 +/- 3 s(-)(1), and was not seen in the absence of GdnHCl.