Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy

Structural studies of the proteins of the BstVI restriction-modification sy stem of Bacillus stearothermophilus V were carried out using intrinsic fluo rescence techniques. The exposure and environments of their tryptophanyl re sidues were determined using collisional quenchers. Quenching of BstVI endo nuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a ra ther exposed tryptophan population. A comparison of the quenching efficienc ies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal ac tivity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ an d Mg2+ binding, with K-d values in the range 3-5 mu M. The binding of S-ade nosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of K-d 550 and 680 mu M at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodid e showed that the presence of S-adenosyl-L-methionine leads to different co nformational states at 20 degrees C and 55 degrees C. These results were in terpreted in terms of differences in the structural characteristics of thes e restriction-modification proteins as well as in terms of differences in t he conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.