1H, 13C, 15N-NMR resonance assignments of oxidized thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii using new methods based on two-dimensional triple-resonance NMR spectroscopy and computer-assisted backbone assignment.
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.
Mittard
V
V
Institut de Biologie Structurale Jean-Pierre Ebel, Commissariat à l'Energie Atomique/Centre National de la Recherche Scientifique, Grenoble, France.
Morelle
N
N
Brutscher
B
B
Simorre
J P
JP
Marion
D
D
Stein
M
M
Jacquot
J P
JP
Lirsac
P N
PN
Lancelin
J M
JM
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000737:chemistry*
D000577:Amides; D054479:Chloroplast Thioredoxins; D011994:Recombinant Proteins; D013879:Thioredoxins