FIGURE 2.

Biophysical characterization of b121a and b122a. A, thermal denaturation of b121a and b122a in a thermal shift assay with Sypro orange dye in PBS, pH 7.4. The fluorescence of the dye in the presence of protein was recorded from 25 to 110 °C, and it was corrected for the corresponding fluorescence of the dye without protein. Both of the proteins show an apparent T_m close to 50 °C. As the protein exposes increased hydrophobic surface with increase in temperature, the dye binds and shows enhanced fluorescence. The data show that b121a and b122a are partially folded and undergo denaturational transitions with temperature. B, analytical gel filtration analysis of b121a (black line) and b122a (blue line) on a Superdex 75 column in PBS buffer at room temperature. For comparison, the elution profile of an equal amount of thioredoxin protein, having a monomer mass of 11.6 kDa is also shown (red line). The absorbance at 220 nm is shown as a function of the elution volume. Both b121a and b122a elute at a similar position as thioredoxin and at the expected position for the monomer. C, FRET experiments demonstrate that b122a is partially structured. A b122a mutant containing a free cysteine proximal to its N terminus was labeled with IAEDANS. Unlabeled protein, labeled protein, and IAEDANS-DTT conjugate were excited at 280 nm, and the emission spectrum was recorded from 300 to 550 nm. The concentrations of proteins (both unlabeled and labeled) used were 20 μm. The excitation and emission slit widths were 2.5 and 5 nm, respectively. Upon the addition of denaturant (guanidine hydrochloride (Gdn)), there is quenching of fluorescence for IAEDANS-DTT conjugate. However, the decrease in fluorescence upon denaturation and reduction is much larger for the labeled protein than for IAEDANS-DTT conjugate. This shows that the energy transfer taking place in the oxidized protein disappears upon denaturation and reduction of the protein disulfides. a.u., arbitrary units.