Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 --> Met145 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 O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 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 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-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 polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.
Federwisch
M
M
Institute of Cell Biology (Cancer Research), University of Essen Medical School, Hufeland-Strasse 55, D-45122 Essen, Germany.
Hassiepen
U
U
Bender
K
K
Dewor
M
M
Rajewsky
M F
MF
Wollmer
A
A
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine; D017931:DNA Primers; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008715:Methionine; D008780:Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan
D017931:DNA Primers; D011994:Recombinant Proteins; D014364:Tryptophan; D008715:Methionine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine