EFFECT OF SINGLE MUTATIONS ON THE STRUCTURAL DYNAMICS OF A DNA-REPAIRENZYME, THE ESCHERICHIA-COLI FORMAMIDOPYRIMIDINE-DNA GLYCOSYLASE - A FLUORESCENCE STUDY USING TRYPTOPHAN RESIDUES AS REPORTER GROUPS

The effects on the structure dynamics of the Escherichia coli wild-typ e formamidopyrimidine-DNA glycosylase (Fpg) protein of the single muta tions Lys57-->Gly (FpgK57G), Pro2-->Gly (FpgP2G) and Pro2-->Glu (FpgP2 E) were studied by fluorescence techniques, namely: lifetime measureme nts and acrylamide quenching of the fluorescence of Trp residues. The fluorescence decays of Fpg and its mutant forms were analysed by the m aximum-entropy method and lifetime distributions in the range 200 ps t o 9 ns were obtained. The lifetime distribution profiles of FpgK57G, F pgP2G and FpgP2E are different from that of wild-type Fpg. Both dynami c and static quenching by acrylamide were observed for all the protein s. At 20 degrees C, the bimolecular collisional quenching rate constan t of the FpgP2E fluorescence by acrylamide was only 0.8 M-1 s(-1) as c ompared to about 1.4 M-1 s(-1) for the three other proteins. At 6 degr ees C, all the spectroscopic properties of these four proteins are abo ut the same. The analysis of experimental data demonstrates that all t hree mutations induce a structural reorganization of the Fpg protein. However, only the P2E mutation lead to a reduced accessibility of some Trp residues to acrylamide quenching. It is concluded that the single P2E replacement induces a conformational change leading to a more rig id globular structure as opposed to the wild type and K57G and P2G mut ations. The influence of the single mutations on the enzyme activities of the Fpg protein is discussed.