Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells

Forlani, Giada; Antwi, Enoch B; Weis, Daniel Georg; Öztürk, Mehmet Ali; Queck, Bastian A W; Brecht, Dominik; Ventura, Barbara Di

doi:10.1021/acssynbio.2c00232

Cited by 3 publications

(1 citation statement)

References 12 publications

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“…Similarly, the slow cycling N414A/V416L mutant shows a somewhat reduced thermal stability with a T m = 48.1 °C, while the slowest N414L/V416L variant seems to be slightly more thermostable ( T m = 60 °C). Thus, while all the tested mutations that tune the recovery of AsLOV2 apparently had some impact on the thermal stability of the protein, the fastest and slowest identified variants should still be stable under moderate cultivation temperatures, e.g., when used in optogenetic experiments (see, e.g., − ) or when employed to generate plants with altered phototropin kinetics …”

Section: Resultsmentioning

confidence: 99%

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime

Hemmer,

Siedhoff,

Werner

et al. 2023

JACS Au

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Naturally occurring and engineered flavin-binding, blue-light-sensing, light, oxygen, voltage (LOV) photoreceptor domains have been used widely to design fluorescent reporters, optogenetic tools, and photosensitizers for the visualization and control of biological processes. In addition, natural LOV photoreceptors with engineered properties were recently employed for optimizing plant biomass production in the framework of a plant-based bioeconomy. Here, the understanding and fine-tuning of LOV photoreceptor (kinetic) properties is instrumental for application. In response to blue-light illumination, LOV domains undergo a cascade of photophysical and photochemical events that yield a transient covalent FMN-cysteine adduct, allowing for signaling. The rate-limiting step of the LOV photocycle is the dark-recovery process, which involves adduct scission and can take between seconds and days. Rational engineering of LOV domains with fine-tuned dark recovery has been challenging due to the lack of a mechanistic model, the long time scale of the process, which hampers atomistic simulations, and a gigantic protein sequence space covering known mutations (combinatorial challenge). To address these issues, we used machine learning (ML) trained on scarce literature data and iteratively generated and implemented experimental data to design LOV variants with faster and slower dark recovery. Over the three prediction–validation cycles, LOV domain variants were successfully predicted, whose adduct-state lifetimes spanned 7 orders of magnitude, yielding optimized tools for synthetic (opto)biology. In summary, our results demonstrate ML as a viable method to guide the design of proteins even with limited experimental data and when no mechanistic model of the underlying physical principles is available.

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Section: Resultsmentioning

confidence: 99%

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime

Hemmer,

Siedhoff,

Werner

et al. 2023

JACS Au

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Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery

Hemmer

Schulte

Knieps-Grünhagen

et al. 2022

Photochem Photobiol Sci

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Light, oxygen, voltage (LOV) photoreceptors are widely distributed throughout all kingdoms of life, and have in recent years, due to their modular nature, been broadly used as sensor domains for the construction of optogenetic tools. For understanding photoreceptor function as well as for optogenetic tool design and fine-tuning, a detailed knowledge of the photophysics, photochemistry, and structural changes underlying the LOV signaling paradigm is instrumental. Mutations that alter the lifetime of the photo-adduct signaling state represent a convenient handle to tune LOV sensor on/off kinetics and, thus, steady-state on/off equilibria of the photoreceptor (or optogenetic switch). Such mutations, however, should ideally only influence sensor kinetics, while being benign with regard to the nature of the structural changes that are induced by illumination, i.e., they should not result in a disruption of signal transduction. In the present study, we identify a conserved hydrophobic pocket for which mutations have a strong impact on the adduct-state lifetime across different LOV photoreceptor families. Using the slow cycling bacterial short LOV photoreceptor PpSB1-LOV, we show that the I48T mutation within this pocket, which accelerates adduct rupture, is otherwise structurally and mechanistically benign, i.e., light-induced structural changes, as probed by NMR spectroscopy and X-ray crystallography, are not altered in the variant. Additional mutations within the pocket of PpSB1-LOV and the introduction of homologous mutations in the LOV photoreceptor YtvA of Bacillus subtilis and the Avena sativa LOV2 domain result in similarly altered kinetics. Given the conserved nature of the corresponding structural region, the here identified mutations should find application in dark-recovery tuning of optogenetic tools and LOV photoreceptors, alike. Graphical abstract

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Optogenetics for sensors: On-demand fluorescent labeling of histone epigenetics

Stepanov,

Zhurlova,

Shuvaeva

et al. 2023

Biochemical and Biophysical Research Communications

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Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells

Cited by 3 publications

References 12 publications

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime

Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery

Optogenetics for sensors: On-demand fluorescent labeling of histone epigenetics

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