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

• 批准号: 8171996
• 负责人: EMINA A STOJKOVIC
• 金额: $ 1.09万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171996
• 关键词: Amino Acid Sequence; Bacteria; Biliverdine; Chicago; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Deinococcus; Engineering; Family; Fluorescence; Funding; Grant; Harvest; Institution; Light; Netherlands; Organism; Photochemistry; Photons; Photoreceptors; Phytochrome; Pigments; Plants; Property; Proteins; Research; Research Personnel; Resources; Rhodopseudomonas; Signaling Protein; Source; Spectrum Analysis; Structure; Tetrapyrroles; Tissues; United States National Institutes of Health; Universities; Work; absorption; base; chromophore; mutant; pi bond; quantum

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 各种生物可以通过称为感光体的大量信号蛋白来感知光。在适当的波长范围内吸收光子后，光感受器会在发色团中发生结构变化，这是一种嵌入蛋白质光感模块中的有机色素。植物色素是最初在植物和细菌中发现的红色光感受器。它们具有独特的能力，可以在两个可视化状态（红光λmax〜700 nm）和PFR（远红色光λmax〜750 nm）之间进行可逆光转换。光激活机制涉及围绕开放链四吡咯本发色团的C15 = C16双键的异构化，从而导致其D形环的翻转。最近，来自Deinococcus radiodurands，DRBPHP的纯植物色素（BPH）已被设计为在哺乳动物组织中用作荧光标记。在与基思·莫法特（Keith Moffat）（芝加哥大学，芝加哥大学）和约翰·肯尼斯（John Kennis）博士（荷兰阿姆斯特丹的Vrije Universiteit）和博士合作，我们确定具有异常光化学的BPH，来自Rhodopseudomonas Palustris的RPBPHP3，P3表示P3的荧光。该BPH调节光收集复合物的合成与第二BPH，RPBPHP2表示P2。 P2和P3具有相同的Biliverdin发色团（BV），并且具有52％的氨基酸序列身份，但它们具有不同的光转化特性。 P2，类似于经典的噬菌体色素，在PR和PFR状态之间交替。 P3是不寻常的，因为它在PR和唯一的PNR（接近红光λmax〜650 nm）状态之间交替。我们确定了通过将超快光谱应用于P2和P3的野生型和突变体的应用来确定荧光和异构化量子产率的因素。这项工作为将BPH基于结构的转化为有效的近红外荧光标记提供了基础。