Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

BRET measurement on CCD camera-based microtiter plate readers.

NanoBRET assays, which utilize bioluminescence energy resonance transfer (BRET), have been widely adopted in drug discovery for measuring both protein-protein interactions and drug target engagement. While the EnVision and other traditional well-scanning plate readers that measure a single well at a time are satisfactory for signal detection for smaller experiments, it becomes challenging to scale these assays to applications that require higher throughput. To address this, we explored the adaptation of the ViewLux and FLIPR plate readers for measuring NanoBRET signal. These plate readers utilize charge-coupled device (CCD) cameras for detection, which enable imaging of the entire assay plate simultaneously. We used tool compounds to generate data from each plate reader and found that the image-based plate readers can be used to measure NanoBRET signals with high S/B and Z´, resulting in comparable IC50 values to those obtained from the EnVision, while requiring less time to complete reads. Consequently, utilization of image-based plate readers for NanoBRET measurement may enable applications that require faster reads, such as for high-throughput screening or kinetics studies.

A light-gated transcriptional recorder for detecting cell-cell contacts.

Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. Here, we report TRACC (Transcriptional Readout Activated by Cell-cell Contacts), a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. TRACC is derived from our previous protein-protein interaction recorders, SPARK (Kim et al., 2017) and SPARK2 (Kim et al., 2019), reported in this journal. TRACC incorporates light gating via the light-oxygen-voltage-sensing (LOV) domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC detects cell-cell contacts with high specificity and sensitivity in mammalian cell culture and that it can be used to interrogate interactions between neurons and glioma, a form of brain cancer.

Deciphering molecular interactions by proximity labeling.

Many biological processes are executed and regulated through the molecular interactions of proteins and nucleic acids. Proximity labeling (PL) is a technology for tagging the endogenous interaction partners of specific protein 'baits', via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. Here we review the development of PL technologies and highlight studies that have applied PL to the discovery and analysis of molecular interactions. In particular, we focus on the use of PL for mapping protein-protein, protein-RNA and protein-DNA interactions in living cells and organisms.

Proximity labeling in mammalian cells with TurboID and split-TurboID.

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein-protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase-related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein-protein interactions or organelle-organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5-7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).

Split-TurboID enables contact-dependent proximity labeling in cells.

Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.

Chaperone mediated detection of small molecule target binding in cells.

The ability to quantitatively measure a small molecule's interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.

Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions.

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested 'AND' gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

A nutrient-induced affinity switch controls mTORC1 activation by its Rag GTPase-Ragulator lysosomal scaffold.

A key step in nutrient sensing is activation of the master growth regulator, mTORC1 kinase, on the lysosomal membrane. Nutrients enable mTORC1 scaffolding by a complex composed of the Rag GTPases (Rags) and Ragulator, but the underlying mechanism of mTORC1 capture is poorly understood. Combining dynamic imaging in cells and reconstituted systems, we uncover an affinity switch that controls mTORC1 lifetime and activation at the lysosome. Nutrients destabilize the Rag-Ragulator interface, causing cycling of the Rags between lysosome-bound Ragulator and the cytoplasm, and rendering mTORC1 capture contingent on simultaneous engagement of two Rag-binding interfaces. Rag GTPase domains trigger cycling by coordinately weakening binding of the C-terminal domains to Ragulator in a nucleotide-controlled manner. Cancer-specific Rag mutants override release from Ragulator and enhance mTORC1 recruitment and signalling output. Cycling in the active state sets the Rags apart from most signalling GTPases, and provides a mechanism to attenuate mTORC1 signalling.