Origins of Fluorescence in Evolved Bacteriophytochromes
Origins of Fluorescence in Evolved Bacteriophytochromes
复制标题
DOI：
10.1074/jbc.m114.589739
复制
发表时间：
2014-11-14
影响因子：
4.8
通讯作者：
Forest, Katrina T.
中科院分区：
文献类型：
--
作者：
Bhattacharya, Shyamosree;Auldridge, Michele E.;Forest, Katrina T.
使用荧光蛋白研究哺乳动物体内过程需要近红外（NIR）生物标志物，其利用该范围内的光穿透组织的能力。细菌光敏色素（BphPs）是一种光感受器，它将近红外光的吸收与光异构化、蛋白质构象变化和信号转导相结合。BphPs已经被工程化以形成NIR荧光团，包括IFP1.4、Wi-Phy和iRFP系列，最初是通过His取代Asp-207。这个位置是暗示性的，因为它的主链羰基是在氢键的距离吡咯环的胆绿素发色团的氮，从而潜在地作为一个关键的瞬态质子汇在光转换。为了解释这些荧光素中荧光的起源，我们解析了IFP1.4和比较非荧光单体光敏色素DrCBD（mon）的晶体结构。IFP1.4中的Met-186和瓦尔-288负责在胆绿素D环周围形成紧密堆积的疏水枢纽。Met-186也主要负责相对于母体BphP的蓝移IFP1.4激发最大值。IFP1.4的结构揭示了侧链取代的直接后果是结构异质性降低和两个表面区域的收缩。出乎意料的是，重新安装了Asp-207的IFP1.4（IFPrev）具有比迄今为止公布的大多数NIR植物荧光剂更高的荧光量子产率（约9%）。在协议中，荧光寿命测量确认异常长的激发态寿命，高达815 ps，在IFP1.4和IFPrev。我们的研究有助于描绘工程BphPs中荧光的起源，并将促进植物荧光素作为生物标志物的广泛采用。
Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBD(mon). Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met-186 is also largely responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp-207 reinstalled (IFPrev) has a higher fluorescence quantum yield (approximate to 9%) than most NIR phytofluors published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFPrev. Our research helps delineate the origin of fluorescence in engineered BphPs and will facilitate the wide-spread adoption of phytofluors as biomarkers.