Structural studies of the proteins of the BstVI restriction- modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 °C and 55 or 60 °C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 °C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with K(d) values in the range 3-5 μM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of K(d) 550 and 680 μM at 20 °C and 55 °C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 °C and 55 °C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 °C and at the temperature at which they are most active.
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