Pratyush Kumar Mishra, Myeong-Gyun Kang, Hakbong Lee, Seungjoon Kim, Subin Choi, Nirmali Sharma, Cheol-Min Park, Jaewon Ko, Changwook Lee, Jeong Kon Seo and Hyun-Woo Rhee
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. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. 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.
        
Representative scheme of Haloalkane_dehalogenase-HLD2 structure and an image from PDBsum server
Databases
PDB-Sum
7WAN Previously Class, Architecture, Topology and Homologous superfamily - PDB-Sum server
FSSP
7WANFold classification based on Structure-Structure alignment of Proteins - FSSP server