Rational Design of Brighter, Monomeric Far-Red Fluorescent Proteins

更明亮的单体远红荧光蛋白的合理设计

• 批准号: 8684957
• 负责人: STEPHEN L. MAYO
• 金额: $ 24.98万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-10 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8684957
• 关键词: Animal Model; Area; Benchmarking; Biodiversity; Biological; Biological Markers; Biological Process; Biology; Chimeric Proteins; Collaborations; Core Protein; Development; Diffusion; Disease; Engineering; Ensure; Equipment; Exhibits; Experimental Designs; Failure; Fluorescence; Generations; Goals; Health; Hemoglobin; Image; Imaging technology; Intuition; Left; Libraries; Life; Light; Medical Research; Mutagenesis; Mutate; N-terminal; Organism; Parents; Positioning Attribute; Procedures; Process; Property; Protein Engineering; Proteins; Recording of previous events; Research; Research Personnel; Side; Site; Skin; Speed; Surface; Tail; Techniques; Technology; Testing; Time; Tissues; Transgenic Organisms; Variant; Vertebrate Biology; Vertebrates; Zebrafish; absorption; base; bioimaging; chromophore; combinatorial; cytotoxic; cytotoxicity; cytotoxicity test; design; directed evolution; grasp; improved; link protein; millimeter; monomer; next generation; novel; phenolate; protein function; public health relevance; red fluorescent protein; research study; screening; tool; trafficking

DESCRIPTION (provided by applicant): The goal of the research proposed here is to create bright, monomeric, far-red fluorescent proteins (FPs) that emit light in the near-infrared (NIR) window. These proteins will represent a new wave of bioimaging agents, with far-reaching implications for medical research and the study of vertebrate biology. A bright FP that can be excited by far-red light and emit light in the NIR would allow researchers to track the expression and localization of proteins in real time within a living organism as NIR light penetrates biologicl tissue with minimal absorption. Traditional FPs emit at shorter wavelengths of light (blue/green/yellow) that are absorbed by biological molecules, notably hemoglobin, and will only penetrate a few millimeters into the skin. There has been great promise with the advent of FP technology and much has been delivered on this, but a bright NIR marker has so far eluded the grasp of traditional engineering efforts. All native red fluorescent proteins (RFPs) are tetramers, whose obligate oligomerization has hampered their usefulness as biological markers. Oligomerization of an FP tag can artificially aggregate its linked protein target, causing abnormalities in localization and even impaired activity. Because a monomeric FP avoids these problems, much effort has gone into engineering monomeric RFP variants. However, every monomeric RFP engineered to date is either dimmer than or blue-shifted from its parent, and is often at least moderately cytotoxic. The failure to deliver a more ideal far-red FP emphasizes a shortcoming in traditional engineering efforts such as directed evolution and site-saturation mutagenesis. What we propose here is the development of a novel technique for monomerizing native RFPs that we expect will improve brightness and minimize cytotoxicity. Using computational protein design (CPD) and high-throughput experimental screening, we will test this process by engineering a bright monomeric variant of the far-red FP HcRed. We will create an ensemble of mutated fluorescent cores by designing important interior structural regions, paving the way for a surface design of residues at the oligomeric interfaces, a process that we have already validated. Finally, to further optimize monomeric variants, we will describe and test standard CPD procedures to optimize brightness, red-shift fluorescence emission, and minimize cytotoxicity. We expect this CPD-driven process to be much more efficient than traditional FP engineering efforts and to produce brighter and less cytotoxic far-red monomeric FPs. To ensure that the designed proteins function in a vertebrate model organism, we will test the cytotoxicity of the protein in zebrafish with the support of Dr. David Prober's lab at Caltech. Our lab is well suited to the proposed research: Dr. Mayo is a world-expert in CPD, we have extensively studied far-red fluorescent proteins, and we have available to us all of the equipment, facilities, and collaboration needed to succeed in our aims.

描述（由申请人提供）：本文提出的研究目标是创建明亮的单体远红荧光蛋白（FP），其在近红外（NIR）窗口中发光。这些蛋白质将代表新一波的生物成像剂，对医学研究和脊椎动物生物学研究具有深远的影响。一个明亮的FP可以被远红光激发并在NIR中发光，这将使研究人员能够在活体内真实的时间内跟踪蛋白质的表达和定位，因为NIR光以最小的吸收穿透生物组织。传统的FP发射较短波长的光（蓝色/绿色/黄色），这些光被生物分子吸收，特别是血红蛋白，并且只能穿透几毫米进入皮肤。FP技术的出现带来了巨大的希望，并且在这方面已经取得了很大进展，但迄今为止，传统的工程技术还无法掌握明亮的近红外标记。所有天然的红色荧光蛋白（RFPs）都是四聚体， 其专性寡聚化阻碍了它们作为生物标记物的有用性。FP标签的寡聚化可以人为地聚集其连接的蛋白质靶标，导致定位异常甚至活性受损。由于单体FP避免了这些问题，因此已经投入了大量精力来设计单体RFP变体。然而，到目前为止，每一个单体RFP都比其亲本更暗或蓝移，并且通常至少具有中度细胞毒性。未能提供更理想的远红FP强调了传统工程努力的缺点，如定向进化和位点饱和诱变。我们在这里提出的是开发一种新的技术，用于单体化天然RFPs，我们希望这将提高亮度并最大限度地减少细胞毒性。使用计算蛋白质设计（CPD）和高通量实验筛选，我们将测试这一过程的工程远红FP HcRed的明亮的单体变体。我们将通过设计重要的内部结构区域来创建突变荧光核心的集合，为寡聚体界面处残基的表面设计铺平道路，这是我们已经验证的过程。最后，为了进一步优化单体变体，我们将描述和测试标准CPD程序，以优化亮度，红移荧光发射，并最大限度地减少细胞毒性。我们预计这种CPD驱动的过程将比传统的FP工程工作更有效，并产生更明亮，细胞毒性更小的远红单体FP。为了确保设计的蛋白质在脊椎动物模式生物中发挥作用，我们将在加州理工学院大卫·普罗伯博士实验室的支持下，在斑马鱼中测试蛋白质的细胞毒性。我们 我们的实验室非常适合这项研究：马约博士是CPD领域的世界级专家，我们广泛研究了远红荧光蛋白，我们拥有实现我们目标所需的所有设备、设施和合作。