Federwisch M, Hassiepen U, Bender K, Dewor M, Rajewsky M F, Wollmer A
Institute of Cell Biology (Cancer Research), University of Essen Medical School, Hufeland-Strasse 55, D-45122 Essen, Germany.
Biochem J. 1997 May 15;324 ( Pt 1)(Pt 1):321-8. doi: 10.1042/bj3240321.
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.
采用等电聚焦、圆二色光谱、稳态荧光光谱和时间分辨荧光光谱，对天然重组人DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）与在半胱氨酸145位点甲基化或苄基化或在活性中心经定点诱变修饰（甲硫氨酸145突变体）的AGT衍生物进行比较。AGT蛋白近似球形，色氨酸残基受到高度限制，但不受二硫键稳定。与天然AGT不同，烷基化的AGT在25℃时沉淀，但在4℃时仍保持单体状态。等电聚焦显示，其pI从8.2（AGT）变为8.4（半胱氨酸145甲基化的AGT）和8.6（半胱氨酸145苄基化的AGT）。甲硫氨酸145突变体的α-螺旋含量降低了约5%，色氨酸残基部分释放。虽然O6-苄基鸟嘌呤的非共价结合未改变AGT的二级结构，但其α-螺旋含量在甲基化时增加了约2%，在苄基化时增加了约4%，总体表明AGT在烷基化过程中发生了微小的构象变化。在AGT中未发现标记其进行多聚泛素化的信号序列。因此，AGT降解的信号仍有待发现。
