Chemistry-based protein labeling in living cells is undoubtedly useful for understanding natural protein functions and for biological/pharmaceutical applications. Here, we report a novel approach for endogenous membrane-bound protein labeling for both in vitro and live cell conditions. A moderately reactive alkyloxyacyl imidazole (AI) assisted by ligand-binding affinity (ligand-directed AI (LDAI)) chemistry allowed us to selectively modify natural proteins, such as dihydrofolate reductase (DHFR) and folate receptor (FR), neither of which could be efficiently labeled using the recently developed ligand-directed tosylate approach. It was clear that LDAI selectively labeled a single Lys(K32) in DHFR, proximal to the ligand-binding pocket. We also demonstrate that the fluorescein-labeled (endogenous, by LDAI) FR works as a fluorescent biosensor on the live KB cell surface, which allowed us to carry out unprecedented in situ kinetic analysis of ligand binding to FR.
Caged enzymes whose activities can be controlled by light represent a powerful tool for various biological analyses. However, limited methods are available for the construction of caged proteins and enzymes. We recently developed a novel protein labeling method termed ligand-directed acyl imidazole (LDAI) chemistry, which allows us to selectively modify natural dihydrofolate reductase and folate receptor in a test tube and in live cell contexts. In this work, we have examined in detail the reaction characteristics of the LDAI chemistry using carbonic anhydrase I (CAI) as a model enzyme. In addition to modifying Lys residues with a carbamate bond, the LDAI method modified Ser and Tyr residues with a carbonate bond. Owing to the relatively labile carbonate bond formed, the LDAI chemistry was demonstrated to be applicable for a rational one-step construction of caged enzymes. This method is simple and based on the transient tethering of an inhibitor with moderate activity that is directed to the active site on an enzyme surface. We successfully showed that the activity of the caged CAI was almost completely suppressed by LDAI-based labeling and fully recovered by photoirradiation in the crude conditions (such as cell lysates) as well as in test tube settings.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.