STRUCTURE-BASED ENGINEERING OF AN EFFICIENT INFRARED FLUORESCENT MARKER

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 各种生物可以通过一大类称为光感受器的信号蛋白来感知光线。当吸收适当波长范围内的光子时，光感受器会发生发色团的结构变化，发色团是嵌入蛋白质感光模块的有机色素。光敏色素是红光感受器，最初在植物中发现，最近在细菌中发现。它们的独特之处在于它们能够在PR(红光~700 nm)和PFR(远红光~750 nm)两个可光异构化状态之间进行可逆光转化。光激活机制涉及开链四吡咯生色团的C15=C16双键附近的异构化，导致其D-环的翻转。最近，耐辐射球菌DrBphP的细菌光敏色素(BPH)已被设计用于哺乳动物组织中的荧光标记。在与芝加哥大学的Keith Moffat博士和荷兰阿姆斯特丹Vrije大学的John Kennis博士的合作下，我们确定沼泽红假单胞菌具有不寻常的光化学RpBphP3的BPH是强荧光的。该BPH与第二个BPH RpBphP2相结合调节捕光复合体的合成，表示为P2。P2和P3具有相同的胆绿素发色团(BV)和52%的氨基酸序列同源性，但它们具有不同的光转化特性。P2类似于经典的细菌光敏色素，在Pr和Pfr状态之间交替。P3是不寻常的，因为它在Pr和独特的Pnr(近红光~650 nm)状态之间交替。我们通过对野生型和突变型P2和P3的超快光谱的应用，确定了决定荧光和异构化量子产率的因素。这项工作为基于结构的BPH转化为高效的近红外荧光标记奠定了基础。通过结构和序列分析的定点突变，我们希望创造P2和P3的突变变体，它们自然比野生型蛋白更具荧光。纯化的蛋白质将通过UV-Vis吸收和荧光光谱进行光转化性质的表征，并进行结晶测试，以便进行X射线衍射实验。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Various organisms can sense light through a large family of signaling proteins known as photoreceptors. Upon absorption of a photon in the appropriate wavelength range photoreceptors undergo structural changes in the chromophore an organic pigment embedded in the photosensory module of the protein. Phytochromes are red-light photoreceptors originally discovered in plants and more recently in bacteria. They are unique in their ability to undergo reversible photoconversion between two photoisomerizable states Pr (red light ~ 700 nm) and Pfr (far-red light ~ 750 nm). The light-activation mechanism involves isomerization around C15=C16 double bond of an open chain tetrapyrrole chromophore resulting in a flip of its D-ring. Recently a bacteriophytochrome (Bph) from Deinococcus radiodurands DrBphP has been engineered for use as a fluorescent marker in mammalian tissues. In collaboration with Dr. Keith Moffat (The University of Chicago Chicago IL) and Dr. John Kennis (Vrije Universiteit Amsterdam Netherlands) we determined that Bph with unusual photochemistry RpBphP3 from Rhodopseudomonas palustris denoted P3 is highly fluorescent. This Bph modulate synthesis of light harvesting complex in combination with a second Bph RpBphP2 denoted P2. P2 and P3 have the same biliverdin chromophore (BV) and share 52% amino acid sequence identity yet they have distinct photoconversion properties. P2 similar to classical bacteriophytochromes alternates between Pr and Pfr states. P3 is unusual since it alternates between Pr and a unique Pnr (near-red light ~ 650 nm) state. We identified factors that determine fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. This work provides the basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker. Through site-directed mutagenesis informed by structural and sequence analysis we want to create mutant variants of P2 and P3 that are naturally more fluorescent than wild-type proteins. Purified proteins will be characterized through UV-vis absorption and fluorescence spectroscopy for photoconversion properties and also tested for crystallization in order to perform X-ray diffraction experiments.