Abstract
Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360 nm cleaves the methyl-6-nitroveratryl core of MeNV-HaXS. MeNV-HaXS covalently links HaloTag- and SNAP-tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV-HaXS-mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV-HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV-HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi-targeted cargo and the assessment of nuclear import kinetics.
Highlights
Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation
MeNV-HaXS was optimized to match the cell permeability of the noncleavable dimerizer of HaloTag and SNAPtag fusion proteins called HaXS8.[16]. Time-dependent dimerization of HaloTag-GFP and SNAP-tag-GFP fusion proteins expressed in HeLa cells was studied in response to the addition of MeNV-HaXS and HaXS8, and we found that MeNV-HaXS and HaXS8 produced dimers at a similar rates
MeNV-HaXS-induced HaloTag-SNAP-tag dimers were stable for > 5 h, and exposure to ambient light did not affect the stability of dimers
Summary
Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Constable University of Basel, Department of Chemistry Spitalstrasse 51, Basel (Switzerland) [+] These authors contributed to this work. We present a novel photocleavable CID, which forms a covalent link between HaloTag-[14] and SNAP-tag[15]-fused proteins.
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