LOV Domains in the Design of Photoresponsive Enzymes

Seifert, Swantje; Brakmann, Susanne

doi:10.1021/acschembio.8b00159

Cited by 21 publications

(18 citation statements)

References 77 publications

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“…Understanding the precise molecular mechanism by which the LOV domain negatively affects T1 tetramerization will require further structural studies. Previous studies on LOV domains have proposed various mechanisms of action, with the one thing in common being rearrangement in LOV secondary structure leading to allosteric effects on the effector domain [43]. With the LOV-Rac1 fusion construct, the LOV domain forms an extensive interface with Rac1 and prevents it from becoming active by an occlusion of the effector binding site of Rac1 [30].…”

Section: Plos Onementioning

confidence: 99%

Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior

et al. 2021

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Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2–3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.

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Section: Plos Onementioning

confidence: 99%

Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior

et al. 2021

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“…We demonstrated with the DTNB assay that H 2 O 2 results in the oxidation of a key cysteine in the iLID protein, which as part of the photoresponse reacts with the flavin cofactor under blue light (Figure S2, Supporting Information). [ 34 ] As a result of the H 2 O 2 treatment iLID no longer bound to Nano under blue light as shown by quartz crystal microbalance with dissipation monitoring (QCM‐D) (Figure S3, Supporting Information). As demonstrated in previous studies the interaction between Ni 2+ ‐NTA and His‐tagged proteins is not affect by H 2 O 2 .…”

Section: Figurementioning

confidence: 99%

Multistimuli Sensing Adhesion Unit for the Self‐Positioning of Minimal Synthetic Cells

Kleineberg

Vidaković‐Koch

et al. 2020

Small

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Cells have the ability to sense different environmental signals and position themselves accordingly in order to support their survival. Introducing analogous capabilities to the bottom‐up assembled minimal synthetic cells is an important step for their autonomy. Here, a minimal synthetic cell which combines a multistimuli sensitive adhesion unit with an energy conversion module is reported, such that it can adhere to places that have the right environmental parameters for ATP production. The multistimuli sensitive adhesion unit senses light, pH, oxidative stress, and the presence of metal ions and can regulate the adhesion of synthetic cells to substrates in response to these stimuli following a chemically coded logic. The adhesion unit is composed of the light and redox responsive protein interaction of iLID and Nano and the pH sensitive and metal ion mediated binding of protein His‐tags to Ni2+‐NTA complexes. Integration of the adhesion unit with a light to ATP conversion module into one synthetic cell allows it to adhere to places under blue light illumination, non‐oxidative conditions, at neutral pH and in the presence of metal ions, which are the right conditions to synthesize ATP. Thus, the multistimuli responsive adhesion unit allows synthetic cells to self‐position and execute their functions.

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“…LOV domains were first discovered in the plant phototropins (phot-1 and phot-2) in Arabidopsis and were determined to be important for phototropism in Arabidopsis (Christie et al, 1998;Christie, Swartz, Bogomolni, & Briggs, 2002;Crosson et al, 2003). Since their discovery, these domains have garnered steady interest, most recently in regard to engineering light-mediated signal transduction processes and optogenetics through the coupling LOV domains to effector domains (Ziegler & Möglich, 2015) and designing photoresponsive molecules (Seifert & Brakmann, 2018).…”

Section: Classes Of Flavoprotein Photoreceptorsmentioning

confidence: 99%

Physical methods for studying flavoprotein photoreceptors

Yee

Chandrasekaran

Lin

et al. 2019

Methods in Enzymology

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Molecular mechanisms of dark-to-light state transitions in flavoprotein photoreceptors have been the subject of intense investigation. Blue-light sensing flavoproteins fall into three general classes that share aspects of their activation processes: LOV domains, BLUF proteins and cryptochromes. In all cases, light-induced changes in flavin redox, protonation and bonding states result in hydrogen-bond and conformational rearrangements important for regulation of downstream targets. Physical characterization of these flavoprotein states can provide valuable insights into biological function, but clear conclusions are often not trivial to obtain owing to challenges in data collection and interpretation. In this chapter, we briefly review the three classes of flavoprotein photoreceptors and provide basic methods for their recombinant production, reconstitution with flavin cofactor, and characterization. We then relate best practices and special considerations for applying several types of spectroscopies, redox potential measurements, and X-ray scattering experiments to this family of flavoprotein. The methods presented are generally accessible to most laboratories.

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LOV Domains in the Design of Photoresponsive Enzymes

Cited by 21 publications

References 77 publications

Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior

Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior

Multistimuli Sensing Adhesion Unit for the Self‐Positioning of Minimal Synthetic Cells

Physical methods for studying flavoprotein photoreceptors

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