Interactive InterPro-based comparisons of proteins in whole genomes.

MOTIVATION: The SWISS-PROT group at the EBI has developed the Proteome Analysis Database utilizing existing resources and providing comprehensive and integrated comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes. The Proteome Analysis Database is accompanied by a program that has been designed to carry out interactive InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes.

The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes (http://www.ebi.ac. uk/proteome/). The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31-67% (InterPro statistics) of the proteins from each of the complete genomes. CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data. The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

NMR solution structure of an oxidised thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii.

NMR solution structures of a cytosolic plant thioredoxin h (112 amino acids, 11.7 kDa) from the green alga Chlamydonmonas reinhardtii have been calculated on the basis of 1904 NMR distance restraints, which include 90 distances used to restrain 45 hydrogen bonds, and 44 phi dihedral restraints. The structure of C. reinhardtii thioredoxin h was solved in its oxidised form, and the ensemble of 23 converged structures superpose to the geometric average structure with an atomic rmsd of 0.080 nm +/- 0.016 for the (N, C(alpha), C) backbone atoms of residues 4-110. Comparisons with other thioredoxins, such as thioredoxin from the bacterium Escherichia coli, thioredoxin 2 from a cyanobacterium of the Anabaena genus, and human thioredoxin, showed that thioredoxin h models share more structural features with human thioredoxin than with other bacterial thioredoxins. Examination of the accessible surface around the redoxactive peptide sequence indicates that a potent thioredoxin-h-substrate interaction could be similar to the vertebrate thioredoxin-substrate interactions.

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties.

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15,101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11,817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.

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.