title: Characterization of thermal stability of the Escherichia coli Fapy-DNA glycosylase.
abstract: Thermal stability of Escherichia coli Fpg protein was studied using far-UV circular dichroism and intrinsic fluorescence. Experimental data indicate that Fpg irreversibly aggregates under heating above 35 degrees C. Heat aggregation is preceded by tertiary conformational changes of Fpg. However, the secondary structure of the fraction that does not aggregate remains unchanged up to approximately 60 degrees C. The kinetics of heat aggregation occurs with an activation enthalpy of approximately 21 kcal/mol. The fraction of monomers forming aggregates decreases with increasing urea concentration, with essentially no aggregation observed above approximately 3 M urea, suggesting that heat aggregation results from hydrophobic association of partially unfolded proteins. With increasing urea concentration, Fpg unfolds in a two-state reversible transition, with a stability of approximately 3.6 kcal/mol at 25 degrees C. An excellent correlation is observed between the unfolded fraction and loss of activity of Fpg. A simple kinetic scheme that describes both the rates and the extent of aggregation at each temperature is presented.
authors.affiliation: Department of Physics (MC 273), Department of Bioengineering (MC 063), University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA. skouznet@uic.edu
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: Laval
authors.forename: A
authors.initials: A
authors.lastname: Ansari
mesh_terms: D002942:Circular Dichroism; D043382:DNA-Formamidopyrimidine Glycosylase; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006358:Hot Temperature; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D014508:Urea; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli