Gooley P R, Caffrey M S, Cusanovich M A, MacKenzie N E
Department of Pharmaceutical Sciences, University of Arizona, Tucson 85721.
Biochemistry. 1990 Mar 6;29(9):2278-90. doi: 10.1021/bi00461a011.
The peptide resonances of the 1H and 15N nuclear magnetic resonance spectra of ferrocytochrome c2 from Rhodobacter capsulatus are sequentially assigned by a combination of 2D 1H-1H and 1H-15N spectroscopy, the latter performed on 15N-enriched protein. Short-range nuclear Overhauser effect (NOE) data show alpha-helices from residues 3-17, 55-65, 69-88, and 103-115. Within the latter two alpha-helices, there are three single 3(10) turns, 70-72, 76-78, and 107-109. In addition alpha H-NHi+1 and alpha H-NHi+2 NOEs indicate that the N-terminal helix (3-17) is distorted. Compared to horse or tuna cytochrome c and cytochrome c2 of Rhodospirillium rubrum, there is a 6-residue insertion at residues 23-29 in R. capsulatus cytochrome c2. The NOE data show that this insertion forms a loop, probably an omega loop. 1H-15N heteronuclear multiple quantum correlation experiments are used to follow NH exchange over a period of 40 h. As the 2D spectra are acquired in short time periods (30 min), rates for intermediate exchanging protons can be measured. Comparison of the NH exchange data for the N-terminal helix of cytochrome c2 of R. capsulatus with the highly homologous horse heart cytochrome c [Wand, A. J., Roder, H., & Englander, S. W. (1986) Biochemistry 25, 1107-1114] shows that this helix is less stable in cytochrome c2.
通过二维¹H-¹H和¹H-¹⁵N光谱相结合的方法，对荚膜红细菌的亚铁细胞色素c₂的¹H和¹⁵N核磁共振谱中的肽共振进行了顺序归属，后者是在¹⁵N富集的蛋白质上进行的。短程核Overhauser效应（NOE）数据显示了残基3 - 17、55 - 65、69 - 88和103 - 115处的α-螺旋。在后两个α-螺旋中，有三个单3(10) 转角，分别位于70 - 72、76 - 78和107 - 109处。此外，αH-NHi+1和αH-NHi+2 NOE表明N端螺旋（3 - 17）发生了扭曲。与马或金枪鱼的细胞色素c以及红螺菌的细胞色素c₂相比，荚膜红细菌细胞色素c₂在残基23 - 29处有一个6个残基的插入。NOE数据表明，这个插入形成了一个环，可能是一个ω环。利用¹H-¹⁵N异核多量子相关实验跟踪了40小时内的NH交换。由于二维谱是在短时间内（30分钟）采集的，因此可以测量中间交换质子的速率。将荚荚荚膜红细菌细胞色素c₂的N端螺旋的NH交换数据与高度同源的马心细胞色素c [Wand, A. J., Roder, H., & Englander, S. W. (1986) Biochemistry 25, 1107 - 1114] 进行比较，结果表明该螺旋在细胞色素c₂中稳定性较差。
