Opaque-2 (O2) is a plant bZIP transcription factor that regulates the expression of alpha and beta prolamines, the main storage proteins in seeds of cereals such as maize and Coix. One of the main processes modulating O2 activity is the heterodimerization with other bZIP transcription factors, but the primary mechanism underlying the partner choice is still unknown. In this paper, we have characterized the bZIP domain of O2 by nuclear magnetic resonance (NMR), circular dichroism (CD), and size-exclusion chromatography. Results obtained from CD measurements suggested that the native O2bZIP has about 40 of its 49 leucine-zipper residues in helical structure, while the DNA-binding domain is completely unstructured. Diffusion-ordered NMR spectroscopy and size-exclusion chromatography showed that O2 forms homodimers in solution. Thermal denaturation experiments indicate that O2 reversibly undergoes dissociation and unfolding in a process that is fully dependent on the protein concentration. Subunit dissociation of O2bZIP dimers, upon dilution of the protein, led to partially folded monomers that retained approximately 80% of the native CD ellipticity at 222 nm. We believe that the existence of partially folded monomers could decrease the entropic penalty for helix formation involved in the DNA binding and in the subunit association of O2bZIP. Stabilization of partially folded monomers may also play a significant role in the dimerization of O2 with other bZIP transcription factors and, consequently, can be important for the regulation of the biological functions of O2 in plants.

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.