STRUCTURE-BASED ENGINEERING OF AN EFFICIENT INFRARED FLUORESCENT MARKER

基于结构的高效红外荧光标记物工程

基本信息

批准号：
8363672
负责人：
EMINA A STOJKOVIC
金额：
$ 1.22万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2012-07-31
项目状态：
已结题

项目摘要

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.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持并且子项目的首席研究员可能是由其他来源提供的，包括其他 NIH 来源。子项目可能列出的总成本代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。各种生物体可以通过一大类被称为光感受器的信号蛋白来感知光。在适当波长范围内吸收光子后，光感受器的发色团发生结构变化，有机色素嵌入蛋白质的感光模块中。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能够在两种光异构化态 Pr（红光 ~ 700 nm）和 Pfr（远红光 ~ 750 nm）之间进行可逆光转换。光激活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 环翻转。最近，来自放射性奇球菌 DrBphP 的细菌光敏色素 (Bph) 已被设计用作哺乳动物组织中的荧光标记。我们与 Keith Moffat 博士（伊利诺伊州芝加哥大学）和 John Kennis 博士（荷兰阿姆斯特丹自由大学）合作，确定来自沼泽红假单胞菌的具有不寻常光化学 RpBphP3 的 Bph（表示为 P3）具有强荧光。该 Bph 与表示为 P2 的第二个 Bph RpBphP2 结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)，并且具有 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 很不寻常，因为它在 Pr 和独特的 Pnr（近红光 ~ 650 nm）状态之间交替。我们通过将超快光谱应用于 P2 和 P3 的野生型和突变体，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 转化为有效的近红外荧光标记物提供了基础。通过结构和序列分析的定点诱变，我们希望创建 P2 和 P3 的突变变体，它们天然比野生型蛋白质更具荧光性。纯化的蛋白质将通过紫外可见吸收和荧光光谱来表征光转换特性，并测试结晶以进行 X 射线衍射实验。