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

Encinas, M., V; SAAVEDRA, C; Vásquez C.

Keywords: magnesium, temperature, sensitivity, spectroscopy, enzyme, fluorescence, protein, structure, tryptophan, conformation, article, iodides, activity, s-adenosylmethionine, type, bacillus, priority, nonhuman, journal, Spectrometry,, ii, Deoxyribonucleases,, Site-Specific, Geobacillus, stearothermophilus, DNA-Methyltransferase, (Adenine-Specific)


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 ?M. 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 ?M 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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Volumen: 263
Número: 1
Editorial: Springer Verlag
Fecha de publicación: 1999
Página de inicio: 65
Página final: 70