Brown M P, Shaikh N, Brenowitz M, Brand L
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
J Biol Chem. 1994 Apr 29;269(17):12600-5.
The Escherichia coli galactose repressor protein (GalR) inhibits transcription of the gal operon upon binding to two operator sites (1-7). This DNA binding activity is inhibited when D-galactose or D-fucose binds to GalR (8-14). Fluorescence spectroscopy was used to characterize the single tryptophan of GalR and to investigate the interaction between galactose and GalR. Fluorescence quenching experiments place both tryptophan residues of the GalR dimer in similar, solvent-exposed locations. Galactose is shown to enhance the intrinsic tryptophan fluorescence of GalR, the source of which is not explained by a change in decay times, but is due to an increase in the pre-exponential factor of the longest of the three fluorescence decay times. It is shown that the beta-anomer of D-galactose is the likely form that binds to GalR. An increase in pH from 6.3 to 9.5 causes the equilibrium association constant (K alpha) describing the galactose-GalR interaction to decrease 10-fold. The interaction is cooperative below pH 9.5. Over the pH range of 6.3 to 9.5, the tryptophan solvent exposure of GalR increases. Galactose binding also induces an increase in exposure. These results, and others presented in this paper, show that both pH and galactose cause global alterations in the structure of GalR.
大肠杆菌半乳糖阻遏蛋白（GalR）与两个操纵位点结合后会抑制半乳糖操纵子的转录（1 - 7）。当D - 半乳糖或D - 岩藻糖与GalR结合时，这种DNA结合活性会受到抑制（8 - 14）。荧光光谱法用于表征GalR的单个色氨酸，并研究半乳糖与GalR之间的相互作用。荧光猝灭实验表明，GalR二聚体的两个色氨酸残基处于相似的、暴露于溶剂的位置。结果显示，半乳糖可增强GalR的固有色氨酸荧光，其来源并非由衰减时间的变化所解释，而是由于三个荧光衰减时间中最长的那个的预指数因子增加所致。研究表明，D - 半乳糖的β - 异头物可能是与GalR结合的形式。pH从6.3升高到9.5会导致描述半乳糖 - GalR相互作用的平衡缔合常数（Kα）降低10倍。在pH 9.5以下，这种相互作用具有协同性。在6.3至9.5的pH范围内，GalR的色氨酸暴露于溶剂的程度增加。半乳糖结合也会导致暴露增加。本文呈现的这些结果以及其他结果表明，pH和半乳糖都会导致GalR结构发生全局性改变。
