Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application-猫眼课题宝

DOI：

10.1021/bi026609p

发表时间：

2002-12-31

期刊：

BIOCHEMISTRY

影响因子：

2.9

通讯作者：

中科院分区：

生物学3区

文献类型：

--

作者：

Hanson, GT;McAnaney, TB;Remington, SJ

新的双发射，pH敏感的变种的绿色荧光蛋白（GFP）已被构建，并适合于在体内的比率发射测量。这类新的GFP，称为deGPFs，是由野生型残基65被苏氨酸取代和残基148和/或203被半胱氨酸取代而产生的。deGFP显示pK（a）值范围为6.8至8.0，发射从绿色形式切换（λ（max）类似于515 nm）的蓝色形式（λ（max）类似于460 nm）与酸化pH值。在这份报告中，我们分析了最详细的deGFP 1变体（S65 T/H148 G/T203 C，pK（a）类似于8.0）和deGFP 4变体（S65 T/C48 S/H148 C/T203 C，pK（a）类似于7.3）。在下面的论文中[McAnaney，T. B.，帕克河，加-地美国，Hanson，G. T.，Remington，S. J.，和Boxer，S. G.（2002）Biochemistry 41，15489-15494]，通过超快荧光上转换光谱法获得的数据可以通过动力学模型来描述，该动力学模型包括在高pH下而不是在低pH下的激发态质子转移途径。进行deGFP 1在高pH和低pH构象下的晶体结构分析以阐明双重发射特性的基础。在低pH下，该结构不包含氢键网络，该氢键网络将支持质子从中性发色团的激发态快速转移到合适的受体;因此观察到蓝色发射。在高pH值下，骨架重排诱导相关的氢键网络的变化允许激发态质子转移从中性发色团的激发态的散装溶剂通过Ser 147和结合水分子，导致绿色发射的阴离子发色团。比较分析表明，双发射的基础是消除野生型质子转移网络的S65 T取代，一般减少氢键的机会，并伴随着增加的疏水性质的发色团环境所产生的半胱氨酸取代。我们通过在PSI 20成纤维细胞中瞬时表达来评估deGFP 4变体用于哺乳动物细胞中细胞内pH测量的适用性。在相同的细胞中比较deGFP 4和市售pH敏感染料SNARF-1对pH变化的响应。结果表明，在检测范围内，两种pH传感器之间的发射率变化的动态范围相当。发现双光子激发与传统的共聚焦显微镜相比，在细胞自发荧光之上引起更好的deGFP 4荧光信号。考虑到它们有利的光学特性，适合生理pH范围的pK（a），以及比率测量的适用性，双发射GFP应该成为研究体内pH的优秀探针。

Novel dual emission, pH-sensitive variants of the green fluorescent protein (GFP) have been constructed and are suitable for ratiometric emission measurements in vivo. This new class of GFPs, termed deGPFs, results from substitution of wild-type residue 65 with threonine and residues 148 and/or 203 with cysteine. deGFPs display pK(a) values ranging from 6.8 to 8.0 and emission that switches from a green form (lambda(max) similar to515 nm) to a blue form (lambda(max) similar to460 nm) with acidifying pH. In this report we analyze in most detail the deGFP1 variant (S65T/H148G/T203C, pK(a) similar to8.0) and the deGFP4 variant (S65T/C48S/ H148C/T203C, pK(a) similar to7.3). In the following paper [McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], data obtained by ultrafast fluorescence upconversion spectroscopy can be described by a kinetic model that includes an excited-state proton-transfer pathway at high pH but not at low pH. Crystal structure analyses of deGFP1 at high-pH and low-pH conformations were performed to elucidate the basis for the dual emission characteristics. At low pH the structure does not contain a hydrogen bond network that would support rapid transfer of a proton from the excited state of the neutral chromophore to a suitable acceptor; hence blue emission is observed. At high pH, backbone rearrangements induced by changes in the associated hydrogen bond network permit excited-state proton transfer from the excited state of the neutral chromophore to the bulk solvent via Ser147 and bound water molecules, resulting in green emission from the anionic chromophore. Comparative analysis suggests that the basis for dual emission is elimination of the wild-type proton-transfer network by the S65T substitution, a general reduction in hydrogen-bonding opportunities, and a concomitant increase in the hydrophobic nature of the chromophore environment resulting from the cysteine substitutions. We evaluated the suitability of the deGFP4 variant for intracellular pH measurements in mammalian cells by transient expression in PSI 20 fibroblasts. The responses of deGFP4 and a commercially available pH-sensitive dye, SNARF-1, to changes in pH were compared in the same cells. Results show that the dynamic range of the emission ratio change is comparable between the two pH sensors over the range examined. Two-photon excitation was found to elicit a better deGFP4 fluorescent signal above cellular autofluorescence when compared to conventional confocal microscopy. Given their favorable optical characteristics, suitable pK(a)'s for the physiological pH range, and suitability for ratiometric measurements, dual emission GFPs should make excellent probes for studying pH in vivo.