Watkins Jennifer L, Kim Hanseong, Markwardt Michele L, Chen Liqing, Fromme Raimund, Rizzo Mark A, Wachter Rebekka M
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
Acta Crystallogr D Biol Crystallogr. 2013 May;69(Pt 5):767-73. doi: 10.1107/S0907444913001546. Epub 2013 Apr 11.
Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.
带有源自色氨酸的发色团的基因编码青色荧光蛋白（CFP）通常在用于活细胞成像应用的Förster共振能量转移（FRET）实验中用作能量供体探针。近年来，人们付出了巨大努力来消除这些蛋白质的结构和激发态异质性，这些异质性与不良的光物理性质有关。最近，增强型CFP的衍生物mCerulean3被引入作为一种优化的FRET供体蛋白，其量子产率高达0.87。在此，报道了mCerulean3的1.6 Å分辨率的X射线结构。发色团在低pH值下呈现平面反式构型，这表明Cerulean的酸诱导异构化已被消除。β链7似乎以单一构象有序排列，这表明发色团附近的构象异质性有所减少。尽管Ile146和Leu167的侧链似乎存在两种旋转异构体状态，但发现它们与发色团的吲哚基团紧密堆积。Ser65回复突变改善了Leu220的侧链堆积。本文还与mTurquoise2进行了结构比较，并讨论了其他工程策略。
