Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy.
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 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 activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees 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 degrees C and at the temperature at which they are most active.
Saavedra
C
C
Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile.
Vásquez
C
C
Encinas
M V
MV
D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007454:Iodides; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000378:metabolism / Q000494:pharmacology; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D007454:Iodides; D012436:S-Adenosylmethionine; D014364:Tryptophan; C057988:DNA modification methylase BstVI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium