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.
An important way to further our understanding of biology and disease is to directly image biological processes as they occur inside a living model organism. This has been a challenge in most vertebrate animals because current imaging technologies, many of which rely on fluorescent protein fusions, cannot easily see past tissue. We propose to engineer a fluorescent protein capable of emitting light of a wavelength to which biological tissue is near-transparent, giving us another tool to further our understanding of the mechanisms involved in health and disease.