Optical Manipulation of Subcellular Protein Translocation Using a Photoactivatable Covalent Labeling System

Abstract

The photoactivatable chemically induced dimerization (photo-CID) technique for tag-fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein-protein interactions.H owever, light-induced covalent protein dimerization in living cells has yet to be established, despite its various advantages.Herein, we developed ap hotoactivatable covalent protein-labeling technology by applying ac aged ligand to the BL-tag system, ac ovalent protein labeling system that uses mutant b-lactamase.Wefurther developed CBHD,acaged protein dimerizer, using caged BL-tag and HaloTag ligands,a nd achieved lightinduced protein translocation from the cytoplasm to subcellular regions.I na ddition, this covalent photo-CID system enabled quickp rotein translocation to al aser-illuminated microregion. These results indicate that the covalent photo-CID system will expand the scope of CID applications in the optical manipulation of cellular functions.

Development of a Versatile Protein Labeling Tool for Live‐Cell Imaging Using Fluorescent β‐Lactamase Inhibitors

“…This labeling system has the advantage of design flexibility that provides cell‐impermeable or cell‐permeable fluorescent probes by modifying the corresponding β‐lactam substrates [11, 13] . These characteristics enable real‐time pulse‐chase analysis, [14] single‐molecule imaging, [15] and optically‐controlled protein dimerization [16] . Meanwhile, the labile β‐lactam ring of the substrates has pronounced chemical susceptibility to acid and alkaline conditions, as well as other nucleophiles, [17] which imposes careful handling for the synthesis and storing of the probes.…”

Quantitative and Repetitive Control of Subcellular Protein–Protein Interaction Using a Photochromic Dimerizer

“…Caged CID systems provide a higher spatiotemporal precision than conventional CID systems. We also developed a caged covalent-CID system that maintains stable protein dimers applicable to gel electrophoresis 26 . These caged CID systems revealed complex cellular systems, such as the specialized properties of a kinesin motor 27 and the molecular mechanisms of spindle asymmetry 28 .…”