Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.
"m being fully understood in all three photoreceptor classes , although some key results emerge to shed light onto the principal mechanisms : More than 10 years ago , ground - breaking results from NMR spectroscopy demonstrated a reordering or even an unfolding of the helix ( named J α ) bridging a LOV2 domain and its signal - transduction module ( Harper et al . , 2004a , b ; Herman et al . , 2013 ) , and thus , displayed a first molecular concept for signal propagation . Subsequent experiments with other LOV domains yielded evidence that also dimerization reactions can occur after light excitation ( Buttani et al . , 2007 ; Möglich and Moffat , 2007 ; Nakasako et al . , 2008 ; Zayner et al . , 2012 ; "
[Show abstract][Hide abstract] ABSTRACT: Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.
Frontiers in Bioscience 10/2015; 2. DOI:10.3389/fmolb.2015.00049 · 3.52 Impact Factor
"ght - oxygen - voltage domains undergo versatile light dependent interactions . In the best - studied LOV domain , LOV2 from Avena sativa phototropin , light - induced thioeither bond formation between a cysteine residue and the FMN chromophore leads to partial unfolding of the C - terminal α - helix ( named Jα ) from the rest of the LOV2 domain ( Harper et al . , 2004 ) . This conformation change has been widely used to construct light - controllable proteins in allosteric or steric manners ( Lee et al . , 2008 ; Strickland et al . , 2008 ; Moglich et al . , 2009 ; Wu et al . , 2009 ; Ohlendorf et al . , 2012 ) . Wu et al . ( 2009 ) constructed photoactivatable small GTPase Rac1 ( PA - Rac1 ; Figure "
[Show abstract][Hide abstract] ABSTRACT: In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.
"Ja-helix displacement therefore represents an integral component of the phototropin light switch. Indeed, artificial disruption of the Ja-helix from the LOV2 core through targeted mutagenesis uncouples this mode of regulation and leads to phot1 activation in the absence of light (Harper et al. 2004, Jones et al. 2007, Kaiserli et al. 2009). "
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