STRUCTURE-BASED ENGINEERING OF AN EFFICIENT INFRARED FLUORESCENT MARKER

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 各种生物都可以通过一个被称为光感受器的信号蛋白大家族来感知光。在吸收适当波长范围内的光子后，光感受器经历发色团的结构变化，发色团是嵌入蛋白质的感光模块中的有机色素。光敏色素是最初在植物中发现的红光光感受器，最近在细菌中发现。它们的独特之处在于它们能够在两种光致异构状态Pr（红光最大值~ 700 nm）和Pfr（远红光最大值~ 750 nm）之间进行可逆的光转换。光活化机制涉及开链四吡咯发色团的C15=C16双键周围的异构化，导致其D-环的翻转。最近，一个细菌光敏色素（Bph）从耐辐射异常球菌，DrBphP，已被工程用作哺乳动物组织中的荧光标记。在与基思莫法特博士（芝加哥大学，芝加哥IL）和约翰Kennis博士（Vrije大学，阿姆斯特丹，荷兰）的合作下，我们确定了具有不寻常的光化学的Bph，来自沼泽红球藻的RpBphP 3，表示为P3，是高度荧光的。该Bph与第二Bph RpBphP 2（表示为P2）组合调节光捕获复合物的合成。P2和P3具有相同的胆绿素发色团（BV），并且共享52%的氨基酸序列同一性，但它们具有不同的光转换特性。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（近红光最大值~ 650 nm）状态之间交替。我们确定的因素，确定荧光和异构化量子产率，通过应用超快光谱的野生型和突变体的P2和P3。这项工作为基于结构的Bph转化为有效的近红外荧光标记物提供了基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 λmax ~ 700 nm) and Pfr (far-red light λmax ~ 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 λmax ~ 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.