Resonance energy transfer sensitises and monitors in situ switching of LOV2-based optogenetic actuators

Abstract

Engineered light-dependent switches provide uniquely powerful opportunities to investigate and control cell regulatory mechanisms. Existing tools offer high spatiotemporal resolution, reversibility and repeatability. Cellular optogenetics applications remain limited with diffusible targets as the response of the actuator is difficult to independently validate. Blue light levels commonly needed for actuation can be cytotoxic, precluding long-term experiments. We describe a simple approach overcoming these obstacles. Resonance energy transfer can be used to constitutively or dynamically modulate actuation sensitivity. This simultaneously offers on-line monitoring of light-dependent switching and precise quantification of activation-relaxation properties in intact living cells. Applying this approach to different LOV2-based switches reveals that flanking sequences can lead to relaxation times up to 11-fold faster than anticipated. In situ–measured parameter values guide the design of target-inhibiting actuation trains with minimal blue-light exposure, and context-based optimisation can increase sensitivity and experimental throughput a further 10-fold without loss of temporal precision.

Light inducible protein degradation inE. coliwith the LOVdeg tag

“…3c). The response is in line with known refolding dynamics of AsLOV2 (Li et al, 2020), where over 50% degradation is only achieved at high frequencies (0.1 seconds -1 ). Next, we tested a LOVdeg tag variant that contains a slowphotocycle mutation, V416I (Zoltowski et al, 2009).…”

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance

“…18 A shortening of the Jα helix as in the psd3 domain or adaptation of the linker region is often necessary to improve an optogenetic construct. 17,26,27 Interestingly, we observed differences in photoreceptor behavior in vitro and in vivo. In vivo, we observed clear differences for the V416L and the I427T variants in the context of the RFP-psd3 constructs, whereas the mutations V416I and V416T did not show an impact on the psd3 module.…”

“…Variations in the linker between the Jα helix and the effector domain are considered critical to optimize the caging of the effector domain in the dark state and to obtain a pronounced light activation. ,, However, our MD simulations indicate that amino acids in this area correlate with amino acids in the core domain and vice versa. Thus, mutations in the core photoreceptor domain can also be used to influence the behavior of the Jα helix in an allosteric way, like the introduction of the V416L mutation into the psd3 domain has shown.…”

“…The iLID mutations diminished the dark-state unfolding of the Jα helix in the AsLOV2 domain and represent an important optimization step of this versatile photoreceptor . A shortening of the Jα helix as in the psd3 domain or adaptation of the linker region is often necessary to improve an optogenetic construct. ,, …”

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

“…Since optogenetic methods are becoming crucial for studying a variety of spatiotemporal properties in signaling networks, they are uniquely positioned to control or modulate time-encoded inputs on cellular targets [135]. Advances such as the establishment of in situ switching of optogenetic actuators based on common blue-light LOV2 photoreceptor rely on engineered resonance energy transfer between two fluorescent proteins [136]. Given the usefulness of optogenetics in addressing fundamental questions about cellular physiology that also factor in calcium signaling, it would be extremely useful to open the possibility of using the most optimized optogenetic tools for one signaling pathway in concert with a Ca 2+ detection method that uses a bioluminescent readout free of crosstalk.…”