Methods to rapidly regulate the levels of proteins in cells are desirable to study the function of proteins of interest. Sequences of amino acids leading to protein degradation (so-called “degrons”) have been discovered, including a four amino acid peptide (RRRG) that leads to a protein's rapid degradation when fused to its C-terminus. Here the authors* describe the construction of a conditional degron where illumination with blue light leads to activation of the degron and degradation of the protein of interest.
To achieve this, a light–oxygen–voltage (LOV) domain, a motif found in plant photoreceptor proteins that have a flavin cofactor absorbing blue light, was employed by engineering a degron into this domain. The flavin cofactor of the LOV domain forms a flavin–cysteine adduct upon exposure to blue light, leading to exposure and unfolding of a C-terminal helix.
The authors reasoned that attaching a RRRG degron to the C-terminus of the LOV domain would lead to conditional degradation of the LOV domain because the C-terminus is buried in the protein core (shielding the degron) until the domain is activated by blue light. This “blue-light inducible degradation” (B-LID) domain can be fused to a protein of interest and protein levels controlled by exposure of cells to nontoxic blue light (465 nm). The RRRG sequence was found to be too potent, and so to achieve a conditional B-LID domain, the less potent RRRGN degron was used. Fusion of the B-LID domain to yellow fluorescent protein (YFP) showed degradation of YFP within in a few hours of exposure to blue light (see Figure).
The authors also showed that this system can work in vivo by injecting mRNA encoding a mCherry-BLID fusion into zebrafish. This work provides a new tool for studying protein function in cells that does not require small molecules or RNAi transfection procedures.