Protein modules that dimerize rapidly upon exposure to light are reported. They permit light-induced control of dimerization of fused protein targets and can be manipulated with two-photon illumination for experiments in thick samples and in vivo. Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase–mediated DNA recombination using light.

Identification of minimal functional CRY2–CIB1 domains and mutations that increased CRY2 photocycle lifetimes combined with the development of an improved photoactivable Cre recombinase enables efficient gene editing. Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein–protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2–CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2–CIB system by identifying versions of CRY2–CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13–α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

Posttranslational modifications of histone proteins regulate gene expression via complex protein–protein and protein–DNA interactions with chromatin. One such modification, the methylation of lysine, has been shown to induce binding to chromodomains in an aromatic cage [Nielsen PR, et al. (2002) Nature 416:103–107]. The binding generally is attributed to the presence of cation–π interactions between the methylated lysine and the aromatic pocket. However, whether the cationic component of the interaction is necessary for binding in the aromatic cage has not been addressed. In this article, the interaction of trimethyllysine with tryptophan is compared with that of its neutral analog, tert-butylnorleucine (2-amino-7,7-dimethyloctanoic acid), within the context of a β-hairpin peptide model system. These two side chains have near-identical size, shape, and polarizabilities but differ in their charges. Comparison of the two peptides reveals that the neutral side chain has no preference for interacting with tryptophan, unlike trimethyllysine, which interacts strongly in a defined geometry. In vitro binding studies of the histone 3A peptide containing trimethyllysine or tert-butylnorleucine to HP1 chromodomain indicate that the cationic moiety is critical for binding in the aromatic cage. This difference in binding affinities demonstrates the necessity of the cation–π interaction to binding with the chromodomain and its role in providing specificity. This article presents an excellent example of synergy between model systems and in vitro studies that allows for the investigation of the key forces that control biomolecular recognition.