Creation of a caspase-3 sensing system using a combination of split-GFP and split-intein.

A genetically encodable caspase-3 sensing system has been created using self-assembling split-GFP, in which a C-terminal fragment is "covalently" cyclized via a caspase-3 substrate sequence mediated by split-intein. The specific cleavage of the cyclic C-terminal fragment by caspase-3 induces the GFP reassembly and fluorescence recovery.

Design and semisynthesis of photoactivable split-GFP by incorporation of photocleavable functionality.

The design of proteins whose structure and function can be manipulated by the external stimuli has been of great interest in the field of protein engineering. In particular, caged proteins which can be activated by photo-irradiation become powerful tools for investigating a variety of biological events. Although protein caging is straightforward to render light-responsive protein functions, this approach mostly have difficulties based on the preparation of caged proteins in which amino acid residues required for biological activities must be specifically modified with synthetic photolabile groups. The synthetic peptide-based strategy for photoactivation of protein function may expand the versatility of protein caging approaches since the photolabile protecting group can be easily introduced into the peptide by means of standard solid-phase methods in a site-specific manner. In this study, we designed a new photoactivable green fluorescent protein (GFP), in which a relatively short C-terminal fragment (residues 214-230) of a dissected protein was modified with 7-diethylamino-4-hydroxymethylcoumarin (DECM) as a photoresponsive-protecting group. The introduced DECM unit completely inhibited the reconstitution with the GFP N-terminal fragment (residues 2-214). However, irradiation of visible light (>400 nm) resulted in efficient cleavage of DECM group, leading to acceleration of protein reassembly and concomitant GFP fluorescence recovery. These results demonstrated direct control of protein structure and function by application of the synthetic photocleavable functionality to a fragmented protein. The combined system of fragmented proteins and synthetic photocleavable elements will provide the useful and potentially wide applicable strategy for the regulation of protein structure and function by the light in a temporal and spacial manner.