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

基本信息

批准号：
9024584
负责人：
Vladislav Verkhusha
金额：
$ 31.73万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9024584
关键词：
Affinity Amino Acids Animals Apoproteins Architecture Bacterial Typing Bilin Biliverdine Binding Biochemical Biology Biophotonics Biosensor Cell Separation Cells Collection Color Detection Development Directed Molecular Evolution Engineering Exhibits Extinction (Psychology)Family Flow Cytometry Fluorescence Fluorescent Probes Green Fluorescent Proteins Health Heme Hemoglobin Image Label Life Light Mammalian Cell Mammals Measurement Melanins Membrane Metabolic Methods Microscopy Modeling Molecular Molecular Evolution Molecular Weight Mus Mutagenesis Optics Oxygenases 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 modality 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 quantum receptor red fluorescent protein response screening spatiotemporal time use tool tumor whole body imaging

项目摘要

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）：对深度组织发育，代谢和致病过程的非侵入性监测将提高现代生物学。在近红外（NIR）透明窗口（NIRW：650-900 nm）中，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光度显着降低，并且吸水吸收性仍然很低。遗传编码的探针中的发色团可以从氨基酸中形成自催化，例如在绿色荧光蛋白（GFP）类似蛋白的情况下，也可以与载脂蛋白结合。类似GFP家族的最红变荧光蛋白（FPS）具有激发和发射光谱，完全或部分在NIRW外部，并且具有低亮度和适度的光稳定性。天然细菌植物色素感受器（BPHP）利用低分子量双膜素作为发色团。 BPHPS结合双脂蛋白比其他含有蛋白质的发色团具有许多优势。首先，与非细菌植物色素的发色团不同，双立替丁在哺乳动物中无处不在。这使BPHP在哺乳动物细胞，组织和整个哺乳动物中的应用与常规GFP样FP一样容易，而无需通过外部溶液提供发色团。其次，BPHP表现出NIR的吸光度和荧光，相对于任何其他植物色素的荧光，它们是红变的，并且位于NIRW内。这使得BPHP频繁地互补与其他现有的生物光电工具（例如所有GFP）（例如FPS和可用的光遗传学工具）。第三，在双列列汀光异构化时，独立的域结构和明显的构象变化使BPHPS有吸引力的模板来设计各种可光活化的NIRFP。根据我们对BPHP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BPHP的NIRFP。其中包括三个明亮和频谱可分离的NIRFP，被推定的短，中和长NIRFP（AIM 1）；具有非光毒性NIR光PA-NIRFP的光活化，最初是黑暗但在短，中或长的NIR光谱区域中变成荧光，并且可以在这些NIR区域之间或反复（rs-nirfps）或反复（RS-NIRFPS）进行摄影（AIM 2）；以及基于可逆的双分子荧光互补方法，利用NIRFP的单体版本的蛋白质相互作用和磷酸化的NIR记者（AIM 3）。我们将基于基于合理结构的设计和候选蛋白的随机诱变采用定向的分子进化方法，然后使用流式细胞仪细菌细胞分类，培养皿上的筛选菌落以及多醇板蛋白质表征。这些常规技术将允许筛选标准的FP特性，例如激发和发射波长，整体亮度，光稳定性，pH稳定性以及在生理温度下的折叠。将开发新的高通量筛选方法，以特别优化基于BPHP的NIRFP。选择具有高量子产率的NIRFP，这是BPHP衍生的FPS的关键参数，将同时使用时间分辨的荧光寿命测量值同时进行。为了筛选不穿透内细菌膜的双核苷的高亲和力，使用血红素加氧酶共表达并将BPHP NIRFPS靶向脉搏 - 将BPHP NIRFPS靶向可用于外源性biliverdin的细菌性周质空间。有希望的NIRFP候选物将使用穿梭载体直接在哺乳动物细胞中筛选，以优化哺乳动物细胞中的蛋白质折叠和稳定性，与内源性双脂蛋白和低细胞毒性相关。优化的NIR探针将在小鼠肿瘤模型中进行测试，并应用于活哺乳动物的研究。所得的NIR探针将将荧光成像方法扩展到体内宏观的深度组织，包括多色细胞和组织标记，细胞光激活和跟踪，酶活性的酶活性和蛋白质相互作用的检测以及哺乳动物组织和整个动物的蛋白质相互作用。