Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein.
Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein.
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DOI：
10.1006/jmbi.2000.3905
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发表时间：
2000-08
影响因子：
5.6
通讯作者：
R. Wachter;D. Yarbrough;K. Kallio;S. Remington
中科院分区：
文献类型：
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作者：
R. Wachter;D. Yarbrough;K. Kallio;S. Remington
黄色荧光蛋白(YFP)的荧光发射已被证明对一些小阴离子(如卤化物)浓度的变化做出快速和可逆的反应，这使得YFP可以用作可遗传编码的氯(-)传感器，可能针对活细胞中的特定细胞器。在阴离子结合时，由于发色团的质子化，荧光被抑制，在低pH值时，相互作用更强。在pH为6.0时，YFP和YFP-H148Q对氯离子的表观解离常数(K(App))分别为32 mm和22 mm，而在pH 7.5时，K(App)分别为777 mm和154 mm。在胞浆中，YFP-H148Q作为卤化物传感器最有希望，因为它对I(-)高度敏感(在pH 7.5时K(App)=23 mM)。为了帮助设计对氯(-)具有更好的特异性和亲和力的变体，我们将YFP-H148Q的apo和I(-)结合的晶体结构解析为2.1A。卤化物结合位在靠近Arg96的一个小的掩埋空腔中，靠近发色团咪唑啉酮氧原子的van der Waals接触处，这提供了静电稳定。卤化物离子与T203Y的苯酚基团氢键，这与突变分析一致，该突变分析表明T203Y是紧密结合所必需的。当阴离子结合时，两亲性部位发生了一系列构象变化，这些变化似乎传播到蛋白质表面148个残基周围的β-凸起区域。阴离子结合提高了生色团的pK(A)值，因为生色团骨架上酚类负电荷的离域被抑制了。阴离子与质子结合平衡的微观结合常数的提取表明，YFP对阴离子的选择性与水合力有关。从尺寸和水化能两个方面提出了改善氯离子与YFP-H148Q结合的具体建议。
The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.