Doug Auld, Ph.D. Novartis Institutes for BioMedical Research
A genetically encoded protein domain that is degraded upon exposure to nontoxic blue light is described in this literature review.
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.
*Abstract from ACS Chemical Biology 2014, Vol. 9: 111–115
Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.
Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in ACS Chemical Biology titled "General method for regulating protein stability with light." Authors of the paper are Bonger KM, Rakhit R, Payumo AY, Chen JK, and Wandless TJ.