The growing family of optogenetic tools seeks to control and monitor cellular activity through light. These genetically encoded proteins have revolutionized neuroscience. Recent advances in the field have discovered a new generation of channelrhodopsin tools (CheRiff, ChrimsonR, and Chronos), whose improved properties have allowed the demonstration of all-optical electrophysiology and multicolor stimulation of a cell population. However, the structural basis of these functional gains is unknown. To date, the C1C2 chimera is the only channelrhodopsin (ChR) with a solved crystal structure. To support increasingly challenging experiments in neurobiology and to facilitate the use of optogenetics in other disciplines, improved channelrhodopsin tools are required. To understand the functional advances discovered in the latest generation of ChRs, structural information is required. I propose to solve a new ChR structure to support and guide efforts in protein engineering. A membrane protein structure is a challenging task, but we have several options and opportunities to overcome common obstacles. In parallel, I will use protein recombination to explore the structure-function relationship of the latest generation of ChRs. Guided by structural information, this SCHEMA method of protein engineering involves swapping blocks of sequence between parental ChRs while minimizing disruption of the tertiary structure. Accurate structural information of a parental ChR will allow for more robust SCHEMA predictions. However, preliminary tests will make use of a homology model (CheRiff model based on C1C2) and a conservative, low- energy chimera library. A representative fraction of this library will be synthesized and transfected into mammalian cells, where several ChR properties will be characterized. This test set will establish links between function and blocks of structure, which will enable modeling and prediction of new block combinations that have desired properties. Throughout the process of recombination we will gain valuable information about structure-function links, and I will produce several functional chimeras. Screening chimeric proteins was a key factor in solving the C1C2 crystal structure, and those chimeras with properties that rival their parents will be added into the structure target pool. Likewise structural information can be used to guide more ambitious chimeragenesis. In this way protein engineering and structural analysis may be used synergistically.
In parallel efforts, I will pursue structural characterization and structure-guided recombination of functionally advanced, optogenetic channelrhodopsins. Recombination (employing a homology model and specialized software to maximize folded and functional chimeras) will link functional traits to blocks of structure. A crystal structure of a parental or chimeric channelrhodopsin will elucidate the structural basis of improved properties and inform future engineering efforts.
| Bedbrook, Claire N; Yang, Kevin K; Rice, Austin J et al. (2017) Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization. PLoS Comput Biol 13:e1005786 |
| Herwig, Lukas; Rice, Austin J; Bedbrook, Claire N et al. (2017) Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore. Cell Chem Biol 24:415-425 |
| Bedbrook, Claire N; Rice, Austin J; Yang, Kevin K et al. (2017) Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins. Proc Natl Acad Sci U S A 114:E2624-E2633 |
