Dataset Information

A Chemical Tool for Blue Light-Inducible Proximity Photo-crosslinking in Live Cells

ABSTRACT: We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Hyun-Woo Rhee

LAB HEAD: Hyun-Woo Rhee

PROVIDER: PXD027149 | Pride | 2022-04-04

REPOSITORIES: Pride

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A chemical tool for blue light-inducible proximity photo-crosslinking in live cells.

Mishra Pratyush Kumar PK Kang Myeong-Gyun MG Lee Hakbong H Kim Seungjoon S Choi Subin S Sharma Nirmali N Park Cheol-Min CM Ko Jaewon J Lee Changwook C Seo Jeong Kon JK Rhee Hyun-Woo HW

Chemical science 20211210 4

We developed a proximity photo-crosslinking method (<i>Spotlight</i>) with a 4-azido-<i>N</i>-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were al ...[more]

PMID: 35211260

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Project description:We have studied the transcriptional, metabolic and photo-physiological responses to light of different spectral quality in the marine diatom Phaeodactylum tricornutum through time-series studies of cultures exposed to equal doses of photosynthetically usable radiation of blue, green and red light. The experiments showed that short-term differences in gene expression and profiles are mainly light quality-dependent. Transcription of photosynthesis-associated nuclear genes was activated mainly through a light quality-independent mechanism likely to rely on chloroplast-to-nucleus signaling. In contrast, genes encoding proteins important for photoprotection and PSII repair were highly dependent on a blue light receptor-mediated signal. Changes in energy transfer efficiency by light-harvesting pigments were spectrally dependent; furthermore, a declining trend in photosynthetic efficiency was observed in red light. The combined results suggest that diatoms possess a light quality-dependent ability to activate photoprotection and efficient repair of photodamaged PSII. In spite of approximately equal numbers of PSII-absorbed quanta in blue, green and red light, the spectral quality of light is important for diatom responses to ambient light conditions. Continuous, axenic culturing of P. tricornutum was done as described in Nymark et al. (2009). The cultures were incubated at 15M-BM-0C under cool white fluorescent light (Philips TLD 36W/96) providing a scalar irradiance (EPAR) of 100 M-NM-<mol m-2 s-1 under continuous white light (CWL) conditions. Upon the onset of the experiment the cultures were synchronized by 48 h dark-treatment (D48). Thereafter the algae were exposed to blue light (BL), green light (GL) or red light (RL) provided by a waveband specific LED panel (SL3500, Photon Systems Instruments). The algae were exposed to 0.5 h, 6 h or 24 h of: 1) 230 M-NM-<mol m-2 s-1 of RL, 2) 100 M-NM-<mol m-2 s-1 of GL, 3) 50 M-NM-<mol m-2 s-1 of BL or 4) 100 M-NM-<mol m-2 s-1 of WL respectively. Three biological replicas for each of the treatments were harvested. Data for the white light exposure and 48h dark treatment is described in GSE42039, PMID: 23520530.

Dataset Information

A Chemical Tool for Blue Light-Inducible Proximity Photo-crosslinking in Live Cells

Publications

A chemical tool for blue light-inducible proximity photo-crosslinking in live cells.

Similar Datasets

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