Caged protein conjugates and light-directed generation of protein activity: preparation, photoactivation, and spectroscopic characterization of caged G-actin conjugates.
A simple method is described to prepare caged (inactive) protein complexes using the amino group-directed photo-deprotection group [(nitroveratryl)oxy]chlorocarbamate (NVOC-Cl). In this study, I show how the polymerization activity of G-actin in physiological salt solution is lost upon conjugation of essential lysine residues of G-actin with NVOC-Cl. Reaction conditions were optimized to prepare caged G-actin in high yield, and the conjugate was characterized by biochemical and absorption spectroscopic methods. Upon excitation of caged G-actin in physiological salt solutions with near-ultraviolet light, an efficient photo-deprotection reaction occurs via photoisomerization of the (nitrophenyl)ethyl group of NVOC, which results in cleavage of the carbamate linkage between the protection reagent and G-actin. A standard irradiation condition was then defined which leads to photoactivation of F-actin from caged G-actin with a yield of more than 90%. Photoactivated F-actin was characterized according to its sedimentation behavior, electron microscopic analysis, and sliding velocity on heavy meromyosin determined with the in vitro motility assay. The results of these assays were similar to those obtained from unmodified F-actin. I also report the preparation of caged G-actin conjugated at cysteine 374 with tetramethylrhodamine iodoacetamide and caged fluorescein maleimide. These caged G-actin conjugates can be used to generate fluorescent, polymerization competent G-actin following near-ultraviolet irradiation. Given the widespread applications of caged substrates and ligands in cell biology, the simple method described herein to prepare and photoactivate caged protein conjugates is expected to advance investigations on the regulation of protein activity in living cells.
Marriott
G
G
Biomolecular and Cellular Dynamics Research Group, Max Planck Institute for Biochemistry, München, Germany.
D000199:Actins / Q000737:chemistry*; D001592:Benzyl Alcohols; D001665:Binding Sites; D002219:Carbamates; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008027:Light; D010782:Photolysis; D011487:Protein Conformation; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D000199:Actins; D001592:Benzyl Alcohols; D002219:Carbamates; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012235:Rhodamines; C088746:((nitroveratryl)oxy)chlorocarbamate; C034996:tetramethylrhodamine iodoacetamide; D003545:Cysteine