LOVTRAP: an optogenetic system for photoinduced protein dissociation

Abstract

Here we introduce LOVTRAP, an optogenetic approach for reversible, light-induced protein dissociation. LOVTRAP is based on protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in Kd. By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins’ access to the cell edge, demonstrating a naturally occurring 3 mHz cell edge oscillation driven by interactions of Vav2, Rac1 and PI3K.

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

“…However, currently all these tools are only bidirectional, either recruiting the protein of interest from the cytoplasm to a specific compartment 4–7,10,15 or translocating it between two different compartments 40 (one in darkness and another in lit conditions). This limitation results from the monochromatic nature of the available protein-targeting tools as they sense light in the same spectral region.…”

“…Later, its natural oligomerization ability was used in optogenetic clustering approaches 9 . Further tuning of the engineered light-activatable systems led to a design of the new generation of dimerizers for advanced control of the protein localization 10 , cell signaling 11 and recombinase activity 12 . All these optogenetic systems sense 440–480 nm light.…”

Engineering genetically-encoded tools for optogenetic control of protein activity

“…Indeed, the application of this approach has been extended to light-inducible activation of protein kinases (Raf1, MEK1, MEK2, and CDK5), using a dark-dimerized Dronpa variant with improved light response [48]. Other photodimerizers showing light-dependent dissociation, such as Zdk, a synthetic protein engineered for selective binding to the dark state of As LOV2, could also be used for similar dark caging strategies [49]. …”

LOVTRAP: A Versatile Method to Control Protein Function with Light

“…As discussed above, the ratio between the two components has different effects in the two strategies described in the Basic protocol. A more quantitative analysis is provided in the original description of LOVTRAP (Wang et al, 2016). Briefly, the concentration of free POI, as a function of LOV:Zdk ratio in the two methods, is given by the following equations: $${\left[POI\right]}_{unbound}=\frac{-(mn-1){\left[POI\right]}_{tot}-Kd+\sqrt{{\{(mn-1){\left[POI\right]}_{tot}+Kd\}}^2+4Kd{\left[POI\right]}_{tot}}}{2}$$$${\left[POI\right]}_{unbound}=\frac{-(m+n-1){\left[POI\right]}_{tot}-Kd+\sqrt{{\{(m+n){\left[POI\right]}_{tot}+Kd\}}^2-4mn{\left[POI\right]}_{tot}^2}}{2}$$ where m is the fraction of LOV2 molecules that are in the closed conformation, n is the ratio of the two components, and Kd is the dissociation constant between Zdk and LOV2 in the closed conformation.…”