Enlightening Allostery: Designing Switchable Proteins by Photoreceptor Fusion

Abstract

upon photoexcitation and often reversible at similar time-scales upon withdrawal of the light trigger. [1][2][3][4] Apart from this temporal precision, light can also be supplied in a spatially confined way with single-cell or subcellular accuracy, for example by using lasers or blinds. [5][6][7] Together, these favorable characteristics render optogenetics a highly powerful method for perturbing and studying dynamic processes in living cells and explain the rapid growth of this scientific field over the past 15 years.Generally, optogenetic tools can either be categorized by the nature of the photoreceptors they employ or by their undelaying working principle. [8] Focusing on the latter classification, one can differentiate between systems that are regulated via inducible association/dissociation reactions and often involve multiple component versus single-component tools functioning via inducible allostery. [8] Association-based tools make use of photoreceptors that bind to one another or to a secondary binding partner upon light exposure. [9] The red/far-red light-responsive PhyB/PIF or blue light-responsive CRY2/CIB1 heterodimerization systems are typical examples for association-dependent optogenetic tools. One application of such optogenetic heterodimerizers is the control of protein subcellular localization. [10] To this end, one binding partner is targeted to a specific subcellular structure or location (e.g., the plasma membrane, nucleus, mitochondria), while the second partner is corecruited to this structure upon dimerization. The same principle can also be employed to reconstitute split-proteins in a light-dependent manner. [9] Optogenetic tools that function via inducible allostery, in contrast, are usually single-component systems. They consist of an engineered chimera between a photoreceptor domain and an effector protein and their light-switching behavior is mediated by steric or allosteric interactions between the two. [8,11] A particular advantage of single-component optogenetic tools as compared to dimerization-based tools is their compactness. They are small in size, since only the photosensory domain is fused to the effector domain/protein of choice and no additional proteins need to be coexpressed. Moreover, allosteric tools do not rely on the diffusion-based association of two components, the kinetics of which depends on the protein concentrations.Optogenetic switches that rely on inducible allostery have mainly been engineered on the basis of light-oxygen-voltage (LOV) domains [11] and phytochromes. [12][13][14] LOV domains belong to the Per-ARNT-Sim or PAS (period circadian protein-aryl Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-componen...

Engineering an Allosteric Control of Protein Function

“…LOV2 consists of a cofactor flavin mononucleotide (FMN), a PAS fold, and a large α-helical region (Jα helix) at the C-terminus of the fold. Upon blue-light exposure, a covalent adduct is formed between the FMN and a cysteine in the PAS fold, resulting in a large conformational change leading to unfolding of the Jα helix. , When irradiation ceases, LOV2 returns to its original conformation within a half-time of ∼30 s. The rapid photocycle and the strong conformational change upon photoexcitation make LOV2 particularly a potent tool . LOV2-based regulations are typically achieved by fusing the C-terminus of Jα to a desired target protein.…”

Combinatorial Approaches for Efficient Design of Photoswitchable Protein-Protein Interactions as In Vivo Actuators

“…Thus, the light-induced partial unfolding of PYP drives the affibody binding to its target. This approach of converting a known binder to opto-binders for the same target is error prone because the photoreceptor insertion tends to change or disrupt the binding to the target even without the allosteric regulation ( Mathony and Niopek, 2021 ). Thus, the current opto-binder design from a known binder is relatively inefficient.…”

Structure‐based design of a photoswitchable affibody scaffold

“…However, altering the target binding loops, the LOV insertion site, or linker length, can weaken, abolish, or in some cases invert coupling 10–12 so that generating a reagent for any new target is likely to involve significant screening of constructs 13 . This unpredictability is not unexpected since the design of complex allosteric coupling in proteins remains a formidable challenge 13,14 . Despite the challenge, significant theoretical and computational progress on allosteric protein design continues to be made 15,16 .…”

“…This approach is particularly powerful because it offers a general method for the design of reversibly photoswitchable affinity reagents that can be encoded in a single construct. However, altering the target binding loops, the LOV insertion site, or linker length, can weaken, abolish, or in some cases invert coupling 10–12 so that generating a reagent for any new target is likely to involve significant screening of constructs 13 . This unpredictability is not unexpected since the design of complex allosteric coupling in proteins remains a formidable challenge 13,14 .…”

“…13 This unpredictability is not unexpected since the design of complex allosteric coupling in proteins remains a formidable challenge. 13,14 Despite the challenge, significant theoretical and computational progress on allosteric protein design continues to be made. 15,16 Design approaches based on a theoretical framework can advance fundamental understanding and offer the possibility of radically focusing the sequence space that must be searched to find variants with ideal function.…”