Mittard V, Morelle N, Brutscher B, Simorre J P, Marion D, Stein M, Jacquot J P, Lirsac P N, Lancelin J M
Institut de Biologie Structurale Jean-Pierre Ebel, Commissariat à l'Energie Atomique/Centre National de la Recherche Scientifique, Grenoble, France.
Eur J Biochem. 1995 Apr 15;229(2):473-85. doi: 10.1111/j.1432-1033.1995.tb20488.x.
The cytosolic thioredoxin h (112 amino acids, 11.8 kDa) cDNA from the eukaryotic green alga Chlamydomonas reinhardtii has been over-expressed in Escherichia coli and the protein was uniformly labelled with 13C and 15N. For the backbone resonance assignments (HN, N, C alpha, CO and H alpha) we used a new set of two-dimensional triple-resonance correlation experiments [Simorre, J.-P., Brutscher, B., Caffrey, M. S. & Marion, D. (1994) J. Biomol. NMR 4, 325-333; Brutscher, B., Simorre, J.-P., Caffrey, M. S. & Marion, D. (1994) J. Magn. Reson. B 105, 77-82] and the new computer assignment protocol ALPS (Assignment for Labelled Protein Spectra) developed in our laboratory [Morelle, N., Brutscher, B., Simorre, J.-P. & Marion, D. (1995) J. Biomol. NMR 5, 154-160]. The assignment was extended to the side chains using three-dimensional HC(C)H-TOCSY correlation experiments together with a set of regular two-dimensional proton spectra. The secondary and super-secondary structures were deduced from the C alpha chemical-shift differences to the random-coil values, the measure of the exchange rates of slow-exchanging amide protons using 15N-HSQC spectroscopy, and the assignment of medium and long-range NOEs. The oxidized C. reinhardtii thioredoxin h is based on an open alpha/beta motif consisting of a five-stranded beta-sheet surrounded by four helices in a manner similar to the bacterial E. coli thioredoxin [Katti, S. K., LeMaster, D. M. & Eklund, H. (1990) J. Mol. Biol. 212, 167-184; Jeng, M.-F., Campbell, A. P., Begley, T., Holmgren, A., Case, D. A., Wright, P. E. & Dyson, H. J. (1994) Structure 2, 853-868] and the human thioredoxin [Qin, J., Clore, G. M. & Gronenborn, A. M. (1994) Structure 2, 503-522] for which C. reinhardtii thioredoxin h shares 32 and 44 sequence identities, respectively. From the analysis of the secondary structure characteristics, the C. reinhardtii thioredoxin h is closer to the human thioredoxin structure than to the bacterial thioredoxin structure. Conversely, the latter would share several structural features with the previously reported [Lancelin, J.-M., Stein, M. & Jacquot, J.-P. (1993) J. Biochem. (Tokyo) 114, 421-431] highly thermostable chloroplast C. reinhardtii thioredoxin m that is involved in the thiol-redox enzymic control of photosynthetic intermediate carbon metabolism.
来自真核绿藻莱茵衣藻的胞质硫氧还蛋白h（112个氨基酸，11.8 kDa）的cDNA已在大肠杆菌中过表达，并且该蛋白用13C和15N进行了均匀标记。对于主链共振归属（HN、N、Cα、CO和Hα），我们使用了一组新的二维三重共振相关实验[Simorre, J.-P., Brutscher, B., Caffrey, M. S. & Marion, D. (1994) J. Biomol. NMR 4, 325 - 333; Brutscher, B., Simorre, J.-P., Caffrey, M. S. & Marion, D. (1994) J. Magn. Reson. B 105, 77 - 82]以及我们实验室开发的新的计算机归属协议ALPS（标记蛋白质谱的归属）[Morelle, N., Brutscher, B., Simorre, J.-P. & Marion, D. (1995) J. Biomol. NMR 5, 154 - 160]。使用三维HC(C)H - TOCSY相关实验以及一组常规二维质子谱将归属扩展到侧链。二级和超二级结构是根据Cα化学位移与无规卷曲值的差异、使用15N - HSQC光谱法测量慢交换酰胺质子的交换率以及中远程和长程NOE的归属推导出来的。氧化型莱茵衣藻硫氧还蛋白h基于一个开放的α/β基序，由一个五链β折叠片层组成，周围环绕着四个螺旋，其方式类似于细菌大肠杆菌硫氧还蛋白[Katti, S. K., LeMaster, D. M. & Eklund, H. (1990) J. Mol. Biol. 212, 167 - 184; Jeng, M.-F., Campbell, A. P., Begley, T., Holmgren, A., Case, D. A., Wright, P. E. & Dyson, H. J. (1994) Structure 2, 853 - 868]以及人硫氧还蛋白[Qin, J., Clore, G. M. & Gronenborn, A. M. (
