Exploring the folding pathways of annexin I, a multidomain protein. I. non-native structures stabilize the partially folded state of the isolated domain 2 of annexin I.

Proteins of the annexin family constitute very attractive models because of their four approximately 70 residue domains, D1 to D4, exhibiting an identical topology comprising five helix segments with only a limited sequence homology of approximately 30%. We focus on the isolated D2 domain, which is only partially folded. A detailed analysis of this equilibrium partially folded state in aqueous solution and micellar solution using 15N-1H multidimensional NMR is presented. Comparison of the residual structure of the entire domain with that of shorter fragments indicates the presence of long-range transient hydrophobic interactions that slightly stabilize the secondary structure elements. The unfolded domain tends to behave as a four-helix, rather than as a five-helix domain. The ensemble of residual structures comprises: (i) a set of native structures consisting of three regions with large helix populations, in rather sharp correspondence with A, B and E helices, and a small helix population in the second part of the C helix; (ii) a set of non-native local structures corresponding to turn-like structures stabilized by several side-chain to side-chain interactions and helix-disruptive side-chains to backbone interactions. Remarkably, residues involved in these local non-native interactions are also involved, in the native structure, in structurally important non-local interactions. During the folding process of annexin I, the local non-native interactions have to switch to native long-range interactions. This structural switch reveals the existence of a sequence-encoded regulation of the folding pathways and kinetics, and emphasizes the key role of the non-native local structures in this regulation.

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.

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.

Secondary structure of the DNA-binding domain of the c-Myb oncoprotein in solution. A multidimensional double and triple heteronuclear NMR study.

The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix-turn-helix (HTH) motif. As a first step towards determination of the three-dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly 15N-labeled and 13C, 15N double-labeled R2R3 and, a selectively 15N-enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 1H, 15N and 13C" atoms and determine the secondary structure of R2R3 in solution. The R3 repeat is composed of three helices (residues 62-75, 78-85 and 91-100) while for the R2 repeat only two helices are found (residues 10-23 and 28-34). The remaining C-terminal part of the R2 repeat, predicted to be helical and part of the HTH motif, undergoes intermediate conformational exchange processes. Stabilization of this segment might occur upon binding to DNA.

Storage and growth of neuroblastoma cells immobilized in calcium-alginate beads.

Mouse neuroblastoma cells (N18) were immobilized in calcium-alginate gel beads. Under standard culture conditions (37 degrees C; 5% CO2), cell growth was observed inside the beads. The number of cells increased threefold during 7 days of culture with cell division and differentiation visualized by electron microscopy. Cell properties maintained after short-term storage (2-3 days at 4 degrees C) included: (i) properties of voltage-dependent ionic channels tested by patch-clamp electrophysiological techniques; (ii) expression of cell-adhesion membrane proteins tested by immunohistochemistry (iii) morphological differentiation obtained by depletion of foetal calf serum in culture medium. The advantages of such an immobilization technique as applied to neurone cells are discussed.

Immobilization of alveolar macrophages for measurement of in vitro dissolution of aerosol particles.

The dissolution of aerosol 57CO3O4 particles was investigated by exposing baboon alveolar macrophages (AM) to the particles and immobilizing the AM in alginate macrobeads crosslinked with Ca2+ ions. AM were obtained from bronchopulmonary lavage and, after immobilization, were stored in culture medium enriched with Ca2+. Dissolution of 57Co3O4 particles by these AM was measured for up to 16 days by gamma counting of filtered media containing beads. For these particles, the daily dissolution rate was 0.242 per cent versus 0.072, 0.089 and 0.091 per cent for beads containing particles only, particles combined with AM enzymes, and non-phagocytic cells, respectively. The rate obtained with immobilized AM is very close to the rates previously found for adherent AM in culture. Moreover, AM that had phagocytosed PuO2 particles before immobilization by the same method gave a mean daily dissolution rate close to that obtained in vivo with baboons. We also demonstrated, by dissolving the alginate network, that immobilization did not affect AM viability. The present method of immobilization seems very promising compared to conventional methods because it facilitates manipulation, allows the use of cells at a high density in a small volume, and gives results for particles with a very low dissolution rate.