Recombinant human O-6-alkylguanine-DNA alkyltransferase (AGT), Cys(145)-alkylated AGT and Cys(145)->Met(145) mutant AGT: Comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy

Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O-6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys(145) or modified by site-directed mutagenesis at the active centre (Met(145) mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8.4 (Cys(145)-methylated AGT) and 8.6 (Cys(145)-benzylated AGT). The alpha-helical content of the Met(145) mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O-6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for poly-ubiquitination. Therefore the signal for AGT degradation remains to be discovered.