Engineering bacterial phytochromes for near-infrared imaging in mammals

用于哺乳动物近红外成像的细菌光敏色素工程

• 批准号: 9220835
• 负责人: Vladislav Verkhusha
• 金额: $ 24.89万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9220835
• 关键词: Affinity; Amino Acids; Animals; Apoproteins; Architecture; Bilin; Biliverdine; Binding; Biochemical; Biology; Biophotonics; Biosensor; Cell Separation; Cells; Collection; Color; Complement; Darkness; Detection; Development; Directed Molecular Evolution; Engineering; Exhibits; Extinction (Psychology); Family; Flow Cytometry; Fluorescence; Fluorescent Probes; Green Fluorescent Proteins; Heme; Hemoglobin; Image; Injectable; Label; Light; Mammalian Cell; Mammals; Measurement; Melanins; Membrane; Metabolic; Methods; Microscopy; Modeling; Modernization; Molecular; Molecular Conformation; Molecular Evolution; Molecular Weight; Mus; Mutagenesis; Optics; Oxygenases; Pathogenicity; Phosphorylation; Photochemistry; Photoreceptors; Physiologic pulse; Physiological; Phytochrome; Process; Production; Property; Protein Engineering; Protein Family; Proteins; Reporter; Shuttle Vectors; Structure; System; Techniques; Technology; Temperature; Testing; Tetrapyrroles; Tissue imaging; Tissues; Water; base; chromophore; cytotoxicity; design; fluorescence imaging; high throughput screening; imaging capabilities; imaging detection; imaging modality; imaging probe; improved; in vivo; in vivo imaging; non-invasive imaging; non-invasive monitor; novel; optical spectra; optogenetics; periplasm; photoactivation; phycobilin; phytochromobilin; protein folding; protein protein interaction; prototype; public health relevance; quantum; receptor; red fluorescent protein; response; screening; spatiotemporal; time use; tool; tumor; whole body imaging

DESCRIPTION (provided by applicant): Non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biology. Imaging of live mammals using fluorescent probes is more feasible within the near-infrared (NIR) transparency window (NIRW: 650-900 nm) where hemoglobin and melanin absorbance significantly decreases and water absorbance is still low. Chromophores in genetically-encoded probes can be formed either autocatalytically from amino acids, as in a case of green fluorescent protein (GFP)-like proteins, or be bound to apoproteins. The most red-shifted fluorescent proteins (FPs) of the GFP-like family have excitation and emission spectra completely or partially outside of the NIRW and suffer from low brightness and modest photostability. Natural bacterial phytochrome photoreceptors (BphPs) utilize low molecular weight biliverdin as a chromophore. BphPs binding biliverdin provide many advantages over other chromophore containing proteins. First, unlike the chromophores of non-bacterial phytochromes, biliverdin is ubiquitous in mammals. This makes BphP applications in mammalian cells, tissues, and whole mammals as easy as conventional GFP-like FPs, without supplying chromophore through an external solution. Second, BphPs exhibit NIR absorbance and fluorescence, which are red-shifted relative to that of any other phytochromes, and lie within the NIRW. This makes BphPs spectrally complementary to other existing biophotonic tools such as all GFP- like FPs and available optogenetic tools. Third, independent domain architecture and pronounced conformational changes upon biliverdin photoisomerization make BphPs attractive templates to design various photoactivatable NIRFPs. Based on our analysis of the photochemistry and structural changes of BphPs we plan to develop three new types of the BphP-based NIRFPs. These include three bright and spectrally resolvable NIRFPs, putatively called short-, medium-, and long-NIRFPs (Aim 1); photoactivatable with non- phototoxic NIR light PA-NIRFPs that are initially dark but become fluorescent either in short-, medium-, or long- NIR spectral regions, and photoswitchable either irreversibly (PS-NIRFPs) or repeatedly (RS-NIRFPs) between these NIR regions (Aim 2); and NIR reporters for protein interactions and phosphorylation based on a reversible bimolecular fluorescence complementation approach utilizing monomerized versions of NIRFPs (Aim 3). We will apply directed molecular evolution approaches based on rational structure-based design and random mutagenesis of candidate proteins, followed by flow cytometry bacterial cell sorting, screening colonies on Petri dishes, and multiwell plate protein characterization. These conventional techniques will allow screening for standard FP properties such as excitation and emission wavelengths, overall brightness, photostability, pH-stability, and folding at physiological temperatures. New high-throughput screening methods will be developed to specifically optimize BphP-based NIRFPs. Selection of NIRFPs with high quantum yield, a crucial parameter for BphP-derived FPs, will be performed using time-resolved fluorescence lifetime measurements of thousands of colonies simultaneously. To screen for a high affinity to biliverdin, which does not penetrate through the inner bacterial membrane, a pulse-chase production of biliverdin using heme oxygenase co-expression and targeting of BphP NIRFPs to bacterial periplasmic space accessible for exogenous biliverdin will be employed. Promising NIRFP candidates will be directly screened in mammalian cells using shuttle vectors to optimize protein folding and stability in mammalian cells, affinity to endogenous biliverdin and low cytotoxicity. Optimized NIR probes will be tested in mouse tumor models and applied to studies in living mammals. The resulting NIR probes will extend fluorescence imaging methods to deep-tissue in vivo macroscopy including multicolor cell and tissue labeling, cell photoactivation and tracking, detection of enzymatic activities and protein interactions in mammalian tissues and whole animals.

描述（由申请人提供）：深层组织发育、代谢和致病过程的非侵入性监测将推动现代生物学的发展。在近红外（NIR）透明窗口（NIRW：650-900 nm）内，使用荧光探针对活哺乳动物进行成像更可行，其中血红蛋白和黑色素吸收显著降低并且水吸收仍然较低。遗传编码的探针中的发色团可以自催化地由氨基酸形成，如在绿色荧光蛋白（GFP）样蛋白的情况下，或者与脱辅基蛋白结合。类GFP家族的大多数红移荧光蛋白（FP）的激发和发射光谱完全或部分在NIRW之外，并且具有低亮度和适度的光稳定性。天然细菌光敏色素光感受器（BphPs）利用低分子量胆绿素作为发色团。结合胆绿素的BphPs提供了许多优于其它含发色团的蛋白质的优点。首先，与非细菌光敏色素的发色团不同，胆绿素在哺乳动物中普遍存在。这使得BphP在哺乳动物细胞、组织和整个哺乳动物中的应用与传统的GFP样FP一样容易，而无需通过外部溶液提供发色团。第二，BphPs表现出近红外吸收和荧光，这是红移相对于任何其他光敏色素，并位于近红外线。这使得BphP在光谱上与其他现有的生物光子工具互补，例如所有GFP样FP和可用的光遗传学工具。第三，独立的结构域架构和显着的构象变化后，胆绿素光异构化BphPs有吸引力的模板，设计各种光活化NIRFPs。基于我们对BphPs的光化学和结构变化的分析，我们计划开发三种新型的基于BphPs的NIRFPs。这些包括三种明亮且光谱可分辨的NIRFPs，通常称为短、中和长NIRFPs（Aim 1）;可被非光毒性NIR光光活化的PA-NIRFPs，其最初是暗的，但在短、中或长NIR光谱区域中变成荧光，并且可被不可逆地光切换或不可逆地光切换。在这些NIR区域之间重复地（PS-NIRFPs）或重复地（RS-NIRFPs）的NIR报告分子（Aim 2）;和基于利用NIRFPs的单体化形式的可逆双分子荧光互补方法的用于蛋白质相互作用和磷酸化的NIR报告分子（Aim 3）。我们将应用基于合理结构设计和随机诱变候选蛋白的定向分子进化方法，然后进行流式细胞术细菌细胞分选，在培养皿上筛选菌落，以及多孔板蛋白表征。这些常规技术将允许筛选标准FP性质，例如激发和发射波长、总体亮度、光稳定性、pH稳定性和在生理温度下的折叠。将开发新的高通量筛选方法，以专门优化基于BphP的NIRFPs。选择具有高量子产率的NIRFPs，BphP衍生的FP的一个关键参数，将使用时间分辨荧光寿命测量数千个菌落同时进行。为了筛选对不穿透细菌内膜的胆绿素的高亲和力，将采用使用血红素加氧酶共表达和BphP NIRFPs靶向外源胆绿素可接近的细菌周质空间的胆绿素的脉冲追踪生产。有前途的NIRFP候选人将直接在哺乳动物细胞中筛选使用穿梭载体，以优化蛋白质折叠和稳定性在哺乳动物细胞中，内源性胆绿素的亲和力和低细胞毒性。优化的近红外探针将在小鼠肿瘤模型中进行测试，并应用于活体哺乳动物的研究。由此产生的近红外探针将荧光成像方法扩展到深层组织的体内宏观检查，包括哺乳动物组织和整个动物中的细胞和组织标记，细胞光活化和跟踪，酶活性和蛋白质相互作用的检测。