Rational Design of Brighter, Monomeric Far-Red Fluorescent Proteins

描述(申请人提供)：这里提出的研究目标是创造在近红外(NIR)窗口发光的明亮的、单体的、远红的荧光蛋白(FP)。这些蛋白质将代表生物显像剂的新浪潮，对医学研究和脊椎动物生物学的研究具有深远的意义。一种明亮的FP可以被远红光激发并在近红外中发光，这将使研究人员能够在近红光穿透生物组织时实时跟踪蛋白质在生物体内的表达和定位，吸收最少。传统的FP发射波长较短的光(蓝/绿/黄)，这些光被生物分子吸收，特别是血红蛋白，只会穿透皮肤几毫米。FP技术的出现带来了巨大的希望，并在这方面取得了很多成果，但到目前为止，一个明亮的近红外标记还没有得到传统工程努力的掌握。所有天然红色荧光蛋白(RFP)都是四聚体， 其专有的寡聚作用阻碍了它们作为生物标记物的用途。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。因为单体FP避免了这些问题，所以很多工作都花在了设计单体RFP变体上。然而，迄今为止设计的每一种单体RFP要么比它的母体更暗，要么比它的母体蓝移，而且通常至少是适度的细胞毒性。未能提供更理想的远红光FP突显了传统工程努力的缺陷，如定向进化和位点饱和突变。我们在这里提出的是开发一种新的技术来单体天然的RFP，我们希望这将提高亮度并将细胞毒性降至最低。利用计算蛋白质设计(CPD)和高通量实验筛选，我们将通过设计远红FP HcRed的明亮单体变体来测试这一过程。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，为低聚物界面残基的表面设计铺平道路，我们已经验证了这一过程。最后，为了进一步优化单体变体，我们将描述和测试标准的CPD程序，以优化亮度、红移荧光发射，并将细胞毒性降至最低。我们希望这种由CPD驱动的过程比传统的FP工程工作更有效率，并生产出更明亮、细胞毒性更低的远红单体FP。为了确保设计的蛋白质在脊椎动物模型生物体中发挥作用，我们将在加州理工大学David Prober博士的实验室的支持下，测试该蛋白质在斑马鱼中的细胞毒性。我们的 Lab非常适合于拟议的研究：Mayo博士是CPD方面的世界专家，我们对远红荧光蛋白进行了广泛的研究，我们拥有实现我们目标所需的所有设备、设施和合作。

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.