Optically Gated Discovery of Protein-Biomolecule Interactions

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The overall goal of research in the Geri lab is to map protein interactomes using discovery technologies that provide orders of magnitude improvements in spatiotemporal resolution over the current state-of-the-art. The motivation for this work is that advancing the resolution of protein interactome discovery technology beyond key milestones, such as single cell and single protein thresholds, will have a field-wide impact analogous to similar advances in transcriptomics and microscopy. The first three years of work in the lab will be focused on creating new technologies by combining photocatalytic proximity labeling, in which light-powered catalysts attached to an affinity handle drive the crosslinking of synthetic affinity probes with nearby proteins, with patterned light and interaction-gated activation to simultaneously enforce multiple dimensions of specificity. The fourth and fifth years of work will focus on applying the mature technologies. The overall strategy is divided along two thrusts, in which labeling specificity is obtained through extrinsic optical control or intrinsic chemical control, and has been designed to be programmatically robust by minimizing project interdependency. Intrinsically selective systems will exploit the high spatial resolution of photocatalytic labeling (5 nm) and use “split” systems that operate when defined protein targets are in proximity. Initial work will use natively expressed orphan peptides as proximity labeling loci to discover their currently unknown receptors. The effort will cover thousands of peptides by using label free ion mobility mass spectrometry for proteomics, maximally leveraged by using optimized labeling probe designs. Split systems will combine multiple photocatalysts targeted to different proteins of interest to make colocalization a dimension of specificity, and will be initially applied to map proteins present at membrane junctions. Extrinsically controlled systems will enable subcellular resolution labeling in human tissue sections and ms-resolution temporal control for the study of transient protein interactions. Both approaches are enabled by combining optical tools for spatiotemporal control of light itself with the total and instantaneously responsive (<1µs) dependence between photocatalytic efficiency and the local supply of light, and each allow for a three order of magnitude increase in resolution vs current tools. Spatially selective labeling will focus on identifying protein interactions unique to cell subpopulations in human tissues, with initial studies focusing on discovering location-conditional interactions driving lymphocyte function in human tonsil and thymus. Later studies will focus on discovering interactome differences between translationally relevant tissue samples. Temporally resolved labeling will combine optogenetic tools and photocatalytic proximity labeling to synchronize and interrogate transient protein interactions. The power of this approach will be fully exploited by studying exocytosis in neurons with millisecond resolution, one of the fastest dynamic biological processes known. Successful development and deployment of these systems for protein interaction discovery will enable the study of large interactome spaces for the first time, and is expected to have a broad impact on the molecular biology community.