Wachter R M, Yarbrough D, Kallio K, Remington S J
Institute of Molecular Biology Departments of Physics and Biology, University of Oregon, Eugene, OR, 97403, USA.
J Mol Biol. 2000 Aug 4;301(1):157-71. doi: 10.1006/jmbi.2000.3905.
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.
黄色荧光蛋白（YFP）的荧光发射已被证明能对某些小阴离子（如卤化物）浓度的变化做出快速且可逆的响应；这使得YFP可作为可基因编码的Cl(-)传感器，用于靶向活细胞中的特定细胞器。由于阴离子结合时发色团的质子化，荧光被抑制，在低pH值下相互作用更强。在pH 6.0时，YFP对Cl(-)的表观解离常数（K(app)）为32 mM，YFP-H148Q为22 mM；而在pH 7.5时，K(app)分别为777 mM和154 mM。在胞质溶胶中，YFP-H148Q作为卤化物传感器似乎最具前景，因为它对I(-)具有高度敏感性（在pH 7.5时K(app)=23 mM）。为了帮助设计对Cl(-)具有更高特异性和亲和力的变体，我们解析了YFP-H148Q的脱辅基和I(-)结合的晶体结构，分辨率达到2.1 Å。卤化物结合位点位于与发色团咪唑啉酮氧原子范德华接触附近，在与Arg96相邻的一个小的埋藏腔中，该腔提供静电稳定作用。卤离子与T203Y的酚基团形成氢键，这与突变分析一致，表明T203Y对于紧密结合是不可或缺的。阴离子结合后两亲位点会发生一系列构象变化，这些变化似乎会传播到蛋白质表面残基148周围的β-凸起区域。阴离子结合会提高发色团的pK(a)值，因为酚盐负电荷在发色团骨架上的离域受到抑制。对阴离子和质子结合的连锁平衡的微观结合常数的提取表明，YFP的阴离子选择性与水合力有关。基于大小和水合能，提出了改善Cl(-)与YFP-H148Q结合的具体建议。
