Griesbeck O, Baird G S, Campbell R E, Zacharias D A, Tsien R Y
Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0647, USA.
J Biol Chem. 2001 Aug 3;276(31):29188-94. doi: 10.1074/jbc.M102815200. Epub 2001 May 31.
Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca(2+)] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.
绿色荧光蛋白（YFP）的黄色突变体是信号转导的基因编码指示剂和监测蛋白质-蛋白质相互作用的融合蛋白的关键组成部分。然而，先前的YFP表现出过度的pH敏感性、氯离子干扰、较差的光稳定性或在37℃时表达不佳。大肠杆菌中的蛋白质进化产生了一种名为柠檬黄（Citrine）的新YFP，其中Q69M突变赋予其比先前YFP低得多的pK(a)（5.7），对氯离子不敏感，光稳定性是先前YFP的两倍，并且在37℃和细胞器中的表达要好得多。卤化物抗性通过柠檬黄的2.2埃X射线晶体结构得到解释，该结构表明甲硫氨酸侧链填充了曾经是与发色团相邻的一个大的卤化物结合腔。在柠檬黄中插入钙调蛋白或融合青色荧光蛋白、钙调蛋白、钙调蛋白结合肽和柠檬黄产生了改进的钙指示剂。这些嵌合体可以靶向多个细胞位置，并首次实现了高尔基体中游离[Ca(2+)]的单细胞成像。除了需要pH或卤化物敏感性的情况外，柠檬黄优于所有先前的YFP，并且在生理信号的基因编码荧光指示剂中特别具有优势。
