6] Light-directed activation of protein activity from caged protein conjugates

Abstract

Publisher Summary This chapter reviews experimental procedures used to prepare and characterize caged reagents and caged protein conjugates of cytoskeleton proteins. The chapter shows that caged proteins can be prepared using both lysine and cysteine-directed caged reagent labeling approaches. The technique will be useful to cage the activity of a large variety of proteins, although one limitation of the lysine-directed labeling strategy is the requirement that wild-type proteins must have a reactive lysine residue in the active site. The cysteine-directed labeling approach has an important advantage in this regard in that standard site-directed mutagenesis techniques can be used to engineer fully active proteins that harbor one or more cysteine residue(s) in the active site. Modification of such critically located cysteine residues with 4,5-dimethoxy-2-nitrobenzyl bromide or other thiol-directed caged reagents will no doubt improve the ease with which the activity of proteins can be caged. Light-directed activation of protein activity may be used to investigate the function of diverse proteins within living cells.