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: 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
• 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
• 来源: https://reporter.nih.gov/project-details/8363672
• 关键词: Amino Acid Sequence; Bacteria; Biliverdine; Chicago; Collaborations; Complex; Crystallization; Deinococcus; Engineering; Family; Fluorescence; Fluorescence Spectroscopy; Funding; Grant; Harvest; Light; National Center for Research Resources; Netherlands; Organism; Photochemistry; Photons; Photoreceptors; Phytochrome; Pigments; Plants; Principal Investigator; Property; Proteins; Research; Research Infrastructure; Resources; Rhodopseudomonas; Sequence Analysis; Signaling Protein; Site-Directed Mutagenesis; Source; Spectrum Analysis; Structure; Testing; Tetrapyrroles; Tissues; United States National Institutes of Health; Universities; Variant; Work; X ray diffraction analysis; X-Ray Diffraction; absorption; base; chromophore; cost; mutant; pi bond; quantum; research study; structural biology

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 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的光子吸收后，光感受器会在发色团中发生结构变化，其中有机色素嵌入了蛋白质的光感模块中。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，可以在两个可拍摄状态（红光〜700 nm）和PFR（远红色光〜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色的C15 = C16双键的异构化，导致其D形环的翻转。最近，已设计了deinocococcus radiodurands drbphp的细菌性乳腺癌（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）（荷兰的Vrije Universiteit）合作的情况下，我们确定来自Rhodopseudomonas Palustris的异常光化学rpbphp3的BPH表示，P3表示P3的含量很高。该BPH调节光收集复合物的合成与第二BPH RPBPHP2相结合。 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PR和PFR状态之间的经典噬菌体色素。 P3是不寻常的，因为它在PR和唯一的PNR（接近红光〜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。通过结构和序列分析的位置定向诱变，我们希望创建P2和P3的突变变体，这些变体自然比野生型蛋白更荧光。纯化的蛋白质将通过紫外线吸收和荧光光谱来表征光转化性能，还测试了结晶以进行X射线衍射实验。