Light Activatable, Fluorescence Reporting cAMP Dependent Protein Kinase

摘要

Light-activatable (also known as "caged") protein is a useful chemical biology tool. It provides a means to control the timing and the location of the protein activation, and potentially allow us to further control the cellular behavior. Fluorescence, on the other hand, is an important readout that is widely used in cellular biology to report the concentration, activity, and location of the protein of interest. In 2004, Muir and his colleagues, using an engineered protein prepared via the expressed protein ligation method, produced a photo-activatable protein with a fluorescent response [1]. In this manuscript, we report a strategy that utilizes active site recognition for propinquity labeling of a non-active site residue. This approach is a more general, and potentially more widely applicable, method for preparing caged and profluorescent proteins. cAMP-dependent protein kinase (PKA), an important signal transduction kinase, was chosen to demonstrate this methodology. The catalytic subunit (C) of PKA contains two readily modifiable Cys residues, one at the base of the active site (Cys-199) and the other approximately 20 A away in the c-term tail region (Cys-343). In the past, PKA catalytic subunit was caged by installing a thiol-reactive caging group directly at Cys 199 in the active site region [2] or at the thiophosphorylated Thr 197 in the activating loop region [3]. Our approach is to selectively conjugate the caging peptide at Cys 343, which has no significant role in structure and activity. After conjugation, this caging peptide can non-covalently block the active site to temporally cease the enzyme activity. The designed caging peptide contains a (i) active site-directed peptide inhibitor to non-covalently block enzyme activity site, (ii) a polyethylene glycol (PEG) spacer to optimally position an appended electrophilic maleimide near Cys-343, and (iii) a tripeptide profluorescent cassette composed of a photolabile (PL) moiety inserted between a fluorophore and a fluorescent quencher to report fluorescence after a successful uncaging [4].