Removal of Chromophore-Proximal Polar Atoms Decreases Water Content and Increases Fluorescence in a Near Infrared Phytofluor
Removal of Chromophore-Proximal Polar Atoms Decreases Water Content and Increases Fluorescence in a Near Infrared Phytofluor
复制标题
DOI：
10.3389/fmolb.2015.00065
复制
发表时间：
2015-11-25
影响因子：
5
通讯作者：
Forest, Katrina T.
中科院分区：
文献类型：
--
作者：
Lehtivuori, Heli;Bhattacharya, Shyamosree;Forest, Katrina T.
基因编码的荧光标记物由于其生物相容性、可控时空表达和光稳定性而彻底改变了细胞和分子生物学。为了在整个动物中实现体内成像，需要更长的激发波长探针，这是由于近红外光穿透组织的上级能力不受来自生物分子的吸收或水的自发荧光的阻碍。荧光素来源于近红外吸收的细菌光敏色素，被设计成在电磁光谱的这一区域发出荧光，尽管高量子产率仍然是一个难以捉摸的目标。一个不变的天冬氨酸残基是最重要的光转换在天然光敏色素，大概是由于其骨架羰基的胆绿素（BV）发色团的吡咯环氮以及侧链的大小和电荷的接近。我们推测，极性相互作用网络形成的带电侧链可能有助于通过质子转移的激发态的衰减。因此，我们选择通过掺入亮氨酸来消除与其羧酸酯侧链极性相互作用的所有可能性，从而进一步探索该氨基酸的作用。所得的荧光蛋白WiPhy2保持BV结合、单体状态和长的最大激发波长，同时使细胞中不期望的原卟啉IX α结合最小化。的晶体结构和时间分辨荧光光谱显示，水附近的BV发色团被排除，从而验证我们的假设，即去除极性相互作用导致增强的荧光，通过增加激发态的寿命。这种新的phytofluor在很宽的pH范围内保持其荧光特性，并且不会受到光漂白的影响。WiPhy2在这类荧光蛋白中实现了迄今为止高荧光量子产率和长照明波长之间的最佳折衷。
Genetically encoded fluorescent markers have revolutionized cell and molecular biology due to their biological compatibility, controllable spatiotemporal expression, and photostability. To achieve in vivo imaging in whole animals, longer excitation wavelength probes are needed due to the superior ability of near infrared light to penetrate tissues unimpeded by absorbance from biomolecules or autofluorescence of water. Derived from near infrared-absorbing bacteriophytochromes, phytofluors are engineered to fluoresce in this region of the electromagnetic spectrum, although high quantum yield remains an elusive goal. An invariant aspartate residue is of utmost importance for photoconversion in native phytochromes, presumably due to the proximity of its backbone carbonyl to the pyrrole ring nitrogens of the biliverdin (BV) chromophore as well as the size and charge of the side chain. We hypothesized that the polar interaction network formed by the charged side chain may contribute to the decay of the excited state via proton transfer. Thus, we chose to further probe the role of this amino acid by removing all possibility for polar interactions with its carboxylate side chain by incorporating leucine instead. The resultant fluorescent protein, WiPhy2, maintains BV binding, monomeric status, and long maximum excitation wavelength while minimizing undesirable protoporphyrin IX alpha binding in cells. A crystal structure and time-resolved fluorescence spectroscopy reveal that water near the BV chromophore is excluded and thus validate our hypothesis that removal of polar interactions leads to enhanced fluorescence by increasing the lifetime of the excited state. This new phytofluor maintains its fluorescent properties over a broad pH range and does not suffer from photobleaching. WiPhy2 achieves the best compromise to date between high fluorescence quantum yield and long illumination wavelength in this class of fluorescent proteins.