title: Effect of single mutations on the structural dynamics of a DNA repair enzyme, the Escherichia coli formamidopyrimidine-DNA glycosylase--a fluorescence study using tryptophan residues as reporter groups.
abstract: The effects on the structure dynamics of the Escherichia coli wild-type formamidopyrimidine-DNA glycosylase (Fpg) protein of the single mutations Lys57-->Gly (FpgK57G), Pro2-->Gly (FpgP2G) and Pro2-->Glu (FpgP2E) were studied by fluorescence techniques, namely: lifetime measurements and acrylamide quenching of the fluorescence of Trp residues. The fluorescence decays of Fpg and its mutant forms were analysed by the maximum-entropy method and lifetime distributions in the range 200 ps to 9 ns were obtained. The lifetime distribution profiles of FpgK57G, FpgP2G and FpgP2E are different from that of wild-type Fpg. Both dynamic and static quenching by acrylamide were observed for all the proteins. At 20 degrees C, the bimolecular collisional quenching rate constant of the FpgP2E fluorescence by acrylamide was only 0.8 M(-1) s(-1) as compared to about 1.4 M(-1) s(-1) for the three other proteins. At 6 degrees C, all the spectroscopic properties of these four proteins are about the same. The analysis of experimental data demonstrates that all three 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 rigid globular structure as opposed to the wild type and K57G and P2G mutations. The influence of the single mutations on the enzyme activities of the Fpg protein is discussed.
authors.affiliation: Muséum National d'Histoire Naturelle, Laboratoire de Photobiologie INSERM U 312, Paris, France.
authors.forename: S V
authors.initials: SV
authors.lastname: Kuznetsov
authors.forename: O M
authors.initials: OM
authors.lastname: Sidorkina
authors.forename: J
authors.initials: J
authors.lastname: Jurado
authors.forename: M
authors.initials: M
authors.lastname: Bazin
authors.forename: P
authors.initials: P
authors.lastname: Tauc
authors.forename: J C
authors.initials: JC
authors.lastname: Brochon
authors.forename: J
authors.initials: J
authors.lastname: Laval
authors.forename: R
authors.initials: R
authors.lastname: Santus
mesh_terms: D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000178:Acrylamides; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D020106:Acrylamide; D014364:Tryptophan; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D011088:DNA Ligases