Photoproximity Profiling of Protein–Protein Interactions in Cells

Abstract

Supplemental Figure 1: Chemical structures of photoproximity probes used in this study. Supplemental Figure 3: Sub-cellular localization and photocleavage with the PhotoPPI system. A) Fluorescence microscopy images of HeLa cells transiently transfected with SNAP-FLAG-NLS, which exhibit strong nuclear localization of the SNAP protein. B) Fluorescence and brightfield microscopy images of HeLa cells transientlytransfected with SNAP-FLAG-NLS show nuclear-localization of FITC signal in cells treated with the non-cleavableFITC-BnG, or PF-BnG in the absence of UV irradiation (left three columns). Progressive exposure to UVirradiation at 365 nm results in complete loss of FITC signal in less than 10 min. Note that DAPI co-staining of the nucleus could not be performed due to overlap in the wavelength for DAPI excitation and nitroveratryl cleavage. C) Magnified view of cells in (B) above and reproduced from Fig. 1C here for comparison. Scale bars in A, B and C equal 8.3, 50 and 8.3 µm, respectively. Supplemental Figure 4: In vitro photoproximity labeling in the presence of whole cell proteome. Anti-biotin (streptavidin-800) and anti-mouse Western blot analyses of PP1 labeled SNAP-FLAG/ a-FLAG antibody complex with and without UV irradiation prior to analysis. Photolabeling was performed in the presence of whole cell lysate. Labels for individual proteins are included at appropriate molecular weights: LC, light chain; HC, heavy chain; "SNAP" label represents SNAP-Tag protein without the FLAG epitope. Supplemental Figure 5: Schematic depicting C-terminal (KEAP1-SNAP) and N-terminal (SNAP-KEAP1) genetic fusions used in photoproximity profiling of KEAP1 in cells. GxS represents a glycine-serine spacer, with X indicating the number of glycines. General synthetic methodsReagents purchased from commercial suppliers were analytical grade and used without further purification. All reactions were carried out in oven dried flasks using anhydrous solvents (Acros) unless otherwise specified. Reaction progress was monitored by thin-layer chromatography on Macherey-Nagel SIL G-25 UV254 TLC plates, visualized with UV light, ceric ammonium molybdate (CAM), p-anisidine, bromophenol blue, 2,4-dinitrophenyl hydrazine (DNP), or KMnO4 TLC stains. Nuclear magnetic resonance spectra were acquired using either a Bruker AVANCE II+ 500; 11.7 Tesla NMR or Bruker DRX 400; 9.3 Tesla NMR instrument. Accurate mass measurements were obtained using an Agilent 6224 Tof-MS instrument. When necessary, compounds were purified via flash column chromatography using Siliaflash F60 60 Å, 230-400 mesh silica gel (Silicycle) Chemical synthesis of photoproximity profiling (PhotoPPI)-probes 4-[[(2,2,2-trifluoroacetyl)amino]methyl]benzoic acid (2)Solid 4-(aminomethyl)benzoic acid 1 (15.1 g, 100 mmol) was dissolved in TFAA (42 mL) cooled to 0 °C. Once dissolved, the ice bath was removed and the reaction was allowed to stir at rt until starting material was consumed, ~2 hr. Upon completion, the reaction was quenched with H2O (100 mL) and precipitate collected via vacuum...

Neighborhood watch: tools for defining locale-dependent subproteomes and their contextual signaling activities

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Structure, Bonding, and Photoaffinity Labeling Applications of Dialkyldiazirines

“…Table 1 provides examples of reagents and conditions that are commonly used for the synthesis of dialkyldiazirines. 12,[62][63][64][65][66][67][68][69][70][71][72][73][74][75] Formation of the imine from 1 is typically carried out using ammonia, either in methanolic solution (entries 5-11) or condensed as a liquid (entries 1-4). For sterically hindered carbonyl substrates, imines can be accessed in two steps via formation of the cyclohexylamine imine/enamine or the O-nitroso oxime before treatment with ammonia (entries 10 and 11).…”

Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors

“…While some of these techniques have been successful, they also come with some significant limitations such as bias against weaker interactions, need for co-factors, and labelling approaches that require initiation by exogenous chemicals which have the potential to unsettle normal cellular homeostasis [95,96]. McCutcheon et al [97] reported a photo-proximity PPI (PhotoPPI) profiling method that allowed the characterization of PPI networking maps in vitro and in live cells. The authors employed a SNAP-tagged protein of interest (POI) and functionalized their DAZ-based probe with a benzylguanine and a photocleavable group (NO 2 -veratryl carbamate), such that the DAZ-functionalized probe was covalently attached to the POI (e.g., redox regulated sensor protein, KEAP1) by the benzylguanine moiety.…”

“…Subsequently, upon irradiation at 365 nm, the NO 2 -veratryl-carbamate group was cleaved off and any proximal proteins to the POI were simultaneously labelled by the DAZ group, thereby identifying any nearby proteins with the POI via a subsequent enrichment and quantitative LC-MS/MS analysis (Figure 16). This technique was used by the authors to elucidate the interactome for KEAP1, validate known interactions, and discover novel ones for KEAP1 with high temporal resolution in live cells [97]. This technique was used by the authors to elucidate the interactome for KEAP1, validate known interactions, and discover novel ones for KEAP1 with high temporal resolution in live cells [97].…”