Lighting the way: Recent insights into the structure and regulation of phototropin blue light receptors
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100594
ISSN1083-351X
AutoresJaynee E. Hart, Kevin H. Gardner,
Tópico(s)Photoreceptor and optogenetics research
ResumoThe phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue-light-sensing Light–Oxygen–Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small-angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the regulatory LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances that have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels. The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue-light-sensing Light–Oxygen–Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small-angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the regulatory LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances that have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels. The phototropin blue light receptors (phots) are unique proteins that have had an outsized impact in the radically different fields of plant physiology and protein engineering. In the former, they are key regulators of growth and photosynthetic competence in plants. Their structure, combining small light-perceiving domains with a catalytic output domain that they control, has also inspired creative applications of the phot light-sensing mechanism to artificially regulate unrelated proteins with blue light via the development of novel genetically encoded optogenetic tools (OTs) (1Losi A. Gardner K.H. Möglich A. Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (119) Google Scholar). Both of these large fields rely on and benefit from accurate information about phototropin regulation and structure: rationally modifying phots can both boost plant growth under low light, while the design and application of OTs rely on detailed knowledge of aspects of phot regulation by blue light. Phototropins are present in both algae and plants (2Li F.W. Rothfels C.J. Melkonian M. Villarreal J.C. Stevenson D.W. Graham S.W. Wong G.K.S. Mathews S. Pryer K.M. The origin and evolution of phototropins.Front. Plant Sci. 2015; 6: 637Crossref PubMed Scopus (53) Google Scholar). In algae, a single phot regulates photoprotection (3Petroutsos D. Tokutsu R. Maruyama S. Flori S. Greiner A. Magneschi L. Cusant L. Kottke T. Mittag M. Hegemann P. Finazzi G. A blue-light photoreceptor mediates the feedback regulation of photosynthesis.Nature. 2016; 537: 563-566Crossref PubMed Scopus (126) Google Scholar), eyespot formation (4Trippens J. Greiner A. Schellwat J. Neukam M. Rottmann T. Lu Y. Kateriya S. Hegemann P. Kreimer G. Phototropin influence on eyespot development and regulation of phototactic behavior in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 4687-4702Crossref PubMed Scopus (41) Google Scholar), and reproduction (5Huang K. Beck C.F. Phototropin is the blue-light receptor that controls multiple steps in the sexual life cycle of the green alga Chlamydomonas reinhardtii.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6269-6274Crossref PubMed Scopus (162) Google Scholar). Due to gene duplication (2Li F.W. Rothfels C.J. Melkonian M. Villarreal J.C. Stevenson D.W. Graham S.W. Wong G.K.S. Mathews S. Pryer K.M. The origin and evolution of phototropins.Front. Plant Sci. 2015; 6: 637Crossref PubMed Scopus (53) Google Scholar), higher plants have two phototropin isoforms, phot1 and phot2, which indirectly influence photosynthesis by altering leaf flatness (6Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6969-6974Crossref PubMed Scopus (573) Google Scholar, 7Sakamoto K. Briggs W.R. Cellular and subcellular localization of phototropin1.Plant Cell. 2002; 14: 1723-1735Crossref PubMed Scopus (335) Google Scholar) and chloroplast positioning (6Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6969-6974Crossref PubMed Scopus (573) Google Scholar, 8Kasahara M. Kagawa T. Oikawa K. Suetsugu N. Miyao M. Wada M. Chloroplast avoidance movement reduces photodamage in plants.Nature. 2002; 420: 829-832Crossref PubMed Scopus (412) Google Scholar), as well as controlling CO2 uptake through stomatal opening (9Kinoshita T. Doi M. Suetsugu N. Kagawa T. Wada M. Shimazaki K.I. Phot1 and phot2 mediate blue light regulation of stomatal opening.Nature. 2001; 414: 656-660Crossref PubMed Scopus (697) Google Scholar). Though phot function has diverged somewhat between these lineages, the underlying structure and activation mechanism are conserved (10Onodera A. Kong S.G. Doi M. Shimazaki K.I. Christie J. Mochizuki N. Nagatani A. Phototropin from Chlamydomonas reinhardtii is functional in Arabidopsis thaliana.Plant Cell Physiol. 2005; 46: 367-374Crossref PubMed Scopus (44) Google Scholar, 11Sullivan S. Petersen J. Blackwood L. Papanatsiou M. Christie J.M. Functional characterization of Ostreococcus tauri phototropin.New Phytol. 2016; 209: 612-623Crossref PubMed Scopus (15) Google Scholar). The model algal phot from Chlamydomonas reinhardtii is somewhat more similar to higher plant phot2 isoforms than phot1, bearing 38% protein sequence identity with phot2 from the model flowering plant Arabidopsis thaliana versus 35% identity with A. thaliana phot1. At a domain level, phots are composed of two light-perceiving Light, Oxygen, or Voltage-sensing (LOV) domains (named LOV1 and LOV2 (12Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (243) Google Scholar)), followed by a serine-threonine kinase domain, which is responsible for propagating the light signal within the cell (12Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (243) Google Scholar, 13Christie J.M. Reymond P. Powell G.K. Bernasconi P. Raibekas A.A. Liscum E. Briggs W.R. Arabidopsis NPH1: A flavoprotein with the properties of a photoreceptor for phototropism.Science. 1998; 282: 1698-1701Crossref PubMed Scopus (515) Google Scholar) (Fig. 1). It is important to note that several classes of proteins with somewhat similar domain structures have been identified outside of photosynthetic organisms, but it is unlikely they are evolutionarily related to the phots. These include bacterial LOV-HK proteins with LOV domains coupled to histidine kinases (14Dikiy I. Edupuganti U.R. Abzalimov R.R. Borbat P.P. Srivastava M. Freed J.H. Gardner K.H. Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 4963-4972Crossref PubMed Scopus (11) Google Scholar, 15Möglich A. Ayers R.A. Moffat K. Design and signaling mechanism of light-regulated histidine kinases.J. Mol. Biol. 2009; 385: 1433-1444Crossref PubMed Scopus (280) Google Scholar) and the fungal and mammalian PAS kinase, which utilizes two Per-ARNT-Sim (PAS) domains (which are a superfamily of environmental sensory domains that include LOV domains) to sense metabolic changes in place of the LOV domains present in phots (16Rutter J. Michnoff C.H. Harper S.M. Gardner K.H. McKnight S.L. PAS kinase: An evolutionarily conserved PAS domain-regulated serine/threonine kinase.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8991-8996Crossref PubMed Scopus (84) Google Scholar). While these proteins all regulate kinase activity by environmental changes sensed by LOV or PAS domains, there are sufficient differences in structure, regulatory details, and origin that we strongly discourage referring to the latter two groups as "phototropin-like" proteins. While the function of phot LOV1 domains remains somewhat unclear (see below), extensive biochemical and biophysical work shows that LOV2s repress kinase activity in darkness, which is released by the light-induced disordering of two alpha helices ("A′α" and "Jα") that flank the LOV2 domain (17Jones M.A. Feeney K.A. Kelly S.M. Christie J.M. Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission.J. Biol. Chem. 2007; 282: 6405-6414Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Harper S.M. Neil L.C. Gardner K.H. Structural basis of a phototropin light switch.Science. 2003; 301: 1541-1544Crossref PubMed Scopus (624) Google Scholar, 19Zayner J.P. Antoniou C. Sosnick T.R. The amino-terminal helix modulates light-activated conformational changes in AsLOV2.J. Mol. Biol. 2012; 419: 61-74Crossref PubMed Scopus (93) Google Scholar, 20Petersen J. Inoue S.I. Kelly S.M. Sullivan S. Kinoshita T. Christie J.M. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.J. Biol. Chem. 2017; 292: 13843-13852Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). This process triggers autophosphorylation of the kinase domain, which is the final step in potentiating phot signaling (13Christie J.M. Reymond P. Powell G.K. Bernasconi P. Raibekas A.A. Liscum E. Briggs W.R. Arabidopsis NPH1: A flavoprotein with the properties of a photoreceptor for phototropism.Science. 1998; 282: 1698-1701Crossref PubMed Scopus (515) Google Scholar). Notably, this light-induced protein unfolding event is not only the linchpin in initiating phot activity, but has also been exploited in the design of a collection of OTs, which regulate diverse cellular phenomena (1Losi A. Gardner K.H. Möglich A. Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (119) Google Scholar), including tracking the animal cardiac pacemaker (21Arrenberg A.B. Stainier D.Y. Baier H. Huisken J. Optogenetic control of cardiac function.Science. 2010; 330: 971-974Crossref PubMed Scopus (345) Google Scholar), regulating cellular mechanosensing (22Valon L. Marín-Llauradó A. Wyatt T. Charras G. Trepat X. Optogenetic control of cellular forces and mechanotransduction.Nat. Commun. 2017; 8: 1-10Crossref PubMed Scopus (113) Google Scholar), and controlling neuronal networks (23Boyden E.S. Zhang F. Bamberg E. Nagel G. Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity.Nat. Neurosci. 2005; 8: 1263-1268Crossref PubMed Scopus (3324) Google Scholar). LOV2-based OTs, while extremely successful on many fronts, are still somewhat limited by the equilibrium between the dark and lit states: there is always residual activity in darkness, and the combination of thermal reversion and inefficiency in allosteric coupling ensures that some molecules spontaneously deactivate in light (18Harper S.M. Neil L.C. Gardner K.H. Structural basis of a phototropin light switch.Science. 2003; 301: 1541-1544Crossref PubMed Scopus (624) Google Scholar, 24Strickland D. Yao X. Gawlak G. Rosen M.K. Gardner K.H. Sosnick T.R. Rationally improving LOV domain–based photoswitches.Nat. Methods. 2010; 7: 623-626Crossref PubMed Scopus (150) Google Scholar). Improving OTs thus benefits from a detailed understanding of LOV2 light activation, particularly regarding how phot LOV2 domains interact with their adjacent A′α and Jα helices and how, in turn, these interact with the kinase domain. While much progress has been made in understanding the photochemistry and early light-induced conformational changes of individual LOV domains, we still have an incomplete understanding of important aspects including structures of the full-length phot proteins and mechanisms linking LOV2 helix release to kinase activation. While recent low-resolution studies have made some inroads, extending these to high resolution has been complicated for us and others in the field by practical issues that likely stem from the multidomain/multilinker architecture of phots and the presence of long activation loops within phot kinase domains (e.g., Nakasako et al. (25Nakasako M. Oide M. Takayama Y. Oroguchi T. Okajima K. Domain organization in plant blue-light receptor phototropin2 of Arabidopsis thaliana studied by small-angle X-ray scattering.Int. J. Mol. Sci. 2020; 21: 1-21Crossref Scopus (1) Google Scholar)). In this review, we will highlight what is known about phot structure and activation, identify outstanding questions in the field, and consider the factors that presently challenge obtaining higher-resolution information on full-length phototropins. LOV domains are members of the PAS domain superfamily that specialize in blue light sensing (12Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (243) Google Scholar, 26Zoltowski B.D. Vaccaro B. Crane B.R. Mechanism-based tuning of a LOV domain photoreceptor.Nat. Chem. Biol. 2009; 5: 827-834Crossref PubMed Scopus (201) Google Scholar, 27Losi A. Polverini E. Quest B. Gärtner W. First evidence for phototropin-related blue-light receptors in prokaryotes.Biophys. J. 2002; 82: 2627-2634Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). They share the canonical PAS domain fold, composed of a five-stranded antiparallel beta sheet with an extended helical connector linking the second and third beta strands (Fig. 2) (18Harper S.M. Neil L.C. Gardner K.H. Structural basis of a phototropin light switch.Science. 2003; 301: 1541-1544Crossref PubMed Scopus (624) Google Scholar, 28Crosson S. Moffat K. Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2995-3000Crossref PubMed Scopus (415) Google Scholar, 29Halavaty A.S. Moffat K. Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana.Acta Crystallogr. F Struct. Biol. Commun. 2013; 69: 1316-1321Crossref Scopus (28) Google Scholar). Many LOV domains contain important N- and C-terminal helical extensions outside of the minimal domain core, including the aforementioned A′α and Jα helices, which play a critical role in phot LOV2 domains by disordering after light excitation as an integral component of the photoactivation process (18Harper S.M. Neil L.C. Gardner K.H. Structural basis of a phototropin light switch.Science. 2003; 301: 1541-1544Crossref PubMed Scopus (624) Google Scholar, 19Zayner J.P. Antoniou C. Sosnick T.R. The amino-terminal helix modulates light-activated conformational changes in AsLOV2.J. Mol. Biol. 2012; 419: 61-74Crossref PubMed Scopus (93) Google Scholar, 20Petersen J. Inoue S.I. Kelly S.M. Sullivan S. Kinoshita T. Christie J.M. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.J. Biol. Chem. 2017; 292: 13843-13852Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 30Harper S.M. Christie J.M. Gardner K.H. Disruption of the LOV-Jα helix interaction activates phototropin kinase activity.Biochemistry. 2004; 43: 16184-16192Crossref PubMed Scopus (248) Google Scholar). In addition to the shared structure of LOV domains, their underlying photochemistry is also well conserved across diverse photoreceptors, including phots (31Glantz S.T. Carpenter E.J. Melkonian M. Gardner K.H. Boyden E.S. Wong G.K.S. Chow B.Y. Functional and topological diversity of LOV domain photoreceptors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E1442-E1451Crossref PubMed Scopus (91) Google Scholar), and more broadly diverse LOV-containing proteins from bacteria (32Jentzsch K. Wirtz A. Circolone F. Drepper T. Losi A. Gärtner W. Jaeger K.E. Krauss U. Mutual exchange of kinetic properties by extended mutagenesis in two short LOV domain proteins from Pseudomonas putida.Biochemistry. 2009; 48: 10321-10333Crossref PubMed Scopus (50) Google Scholar), fungi (26Zoltowski B.D. Vaccaro B. Crane B.R. Mechanism-based tuning of a LOV domain photoreceptor.Nat. Chem. Biol. 2009; 5: 827-834Crossref PubMed Scopus (201) Google Scholar), and plants (33Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (250) Google Scholar). Central to this process is a flavin chromophore, most commonly flavin mononucleotide (FMN) but occasionally flavin adenine dinucleotide (FAD) (34Schwerdtfeger C. Linden H. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation.EMBO J. 2003; 22: 4846-4855Crossref PubMed Scopus (232) Google Scholar, 35He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. White collar-1, a DNA binding transcription factor and a light sensor.Science. 2002; 297: 840-843Crossref PubMed Scopus (342) Google Scholar) or riboflavin (36Rivera-Cancel G. Ko W.H. Tomchick D.R. Correa F. Gardner K.H. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 17839-17844Crossref PubMed Scopus (59) Google Scholar) in certain proteins. While the flavin chromophore is noncovalently bound in darkness, blue light excitation triggers the formation of a covalent photoadduct between the isoalloxazine C4(a) atom and a conserved cysteine residue (C512 in Arabidopsis phot1 LOV2 "AtLOV2"; Fig. 2). Concomitantly, the isoalloxazine N5 position becomes protonated, triggering hydrogen bonding changes to an adjacent glutamine residue (Q575) (37Nozaki D. Iwata T. Ishikawa T. Todo T. Tokutomi S. Kandori H. Role of Gln1029 in the photoactivation processes of the LOV2 domain in Adiantum phytochrome3.Biochemistry. 2004; 43: 8373-8379Crossref PubMed Scopus (99) Google Scholar, 38Nash A.I. Ko W.H. Harper S.M. Gardner K.H. A conserved glutamine plays a central role in LOV domain signal transmission and its duration.Biochemistry. 2008; 47: 13842-13849Crossref PubMed Scopus (83) Google Scholar). This change is thought to propagate from this glutamine within the flavin-binding pocket to the LOV domain surface; while details of subsequent steps diverge among LOV proteins (1Losi A. Gardner K.H. Möglich A. Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (119) Google Scholar), for phot LOV2 domains this leads to unfolding of the A′α and Jα helices and activation of kinase activity (37Nozaki D. Iwata T. Ishikawa T. Todo T. Tokutomi S. Kandori H. Role of Gln1029 in the photoactivation processes of the LOV2 domain in Adiantum phytochrome3.Biochemistry. 2004; 43: 8373-8379Crossref PubMed Scopus (99) Google Scholar, 38Nash A.I. Ko W.H. Harper S.M. Gardner K.H. A conserved glutamine plays a central role in LOV domain signal transmission and its duration.Biochemistry. 2008; 47: 13842-13849Crossref PubMed Scopus (83) Google Scholar, 39Iuliano J.N. Collado J.T. Gil A.A. Ravindran P.V. Lukacs A. Shin S. Woroniecka H.A. Adamczyk K. Aramini J.M. Edupuganti U.R. Hall C.R. Unraveling the mechanism of a LOV domain optogenetic sensor: A glutamine lever induces unfolding of the Jα helix.ACS Chem. Biol. 2020; 15: 2752-2765Crossref PubMed Scopus (14) Google Scholar). After illumination ends, the cysteinyl photoadduct decays on the timescale of seconds to hours (e.g., t1/2=29 s for AsPhot1 (Avena sativa) LOV2 at room temperature (33Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (250) Google Scholar)), returning the domain to its original noncovalent chromophore and folded structural state, thus completing the photocycle that governs the activity of LOV-based photoreceptors. Interestingly, while the basic characteristics of the photocycle itself are conserved, activated state lifetimes and quantum efficiencies vary substantially among LOV domains in different photoreceptors. Some LOV domains very slowly recover to the dark state: the best-studied example is the fungal photoreceptor VVD, which has a half-lifetime of dark state reversion of 2.5 h (26Zoltowski B.D. Vaccaro B. Crane B.R. Mechanism-based tuning of a LOV domain photoreceptor.Nat. Chem. Biol. 2009; 5: 827-834Crossref PubMed Scopus (201) Google Scholar). Phot LOV2 domains, by contrast, recover to the dark state relatively quickly (33Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (250) Google Scholar). This fast recovery can limit the light sensitivity and signaling efficiency of both phots and LOV2-based optogenetic tools (1Losi A. Gardner K.H. Möglich A. Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (119) Google Scholar), although this feature also allows such tools to be used in studies of relatively short timescale biological phenomena. Turning to quantum efficiency (QE), phot LOV2 domains tend to have higher QEs than LOV1s although this varies by source: LOV2s from higher plant phot1s are tenfold more efficient than LOV1s (33Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (250) Google Scholar, 40Salomon M. Christie J.M. Knieb E. Lempert U. Briggs W.R. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.Biochemistry. 2000; 39: 9401-9410Crossref PubMed Scopus (508) Google Scholar), dropping to twofold more efficient in the Chlamydomonas phot and higher plant phot2s (33Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (250) Google Scholar). Combined with differences in photocycle length, phot1 dominates phot2 in most responses in higher plants (6Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6969-6974Crossref PubMed Scopus (573) Google Scholar, 12Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (243) Google Scholar, 33Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (250) Google Scholar). Though united by the same overall structure and mechanism, these differences highlight the functional impact of differences in light sensitivity and quantum efficiency between LOV-containing photoreceptors. Given this impact on photobiology coupled with engineered applications with OTs, LOV domain photocycles have been extensively studied and modified through random (41Christie J.M. Corchnoy S.B. Swartz T.E. Hokenson M. Han I.S. Briggs W.R. Bogomolni R.A. Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1.Biochemistry. 2007; 46: 9310-9319Crossref PubMed Scopus (83) Google Scholar, 42Kawano F. Aono Y. Suzuki H. Sato M. Fluorescence imaging-based high-throughput screening of fast-and slow-cycling LOV proteins.PLoS One. 2013; 8e82693Crossref PubMed Scopus (46) Google Scholar) and rational (26Zoltowski B.D. Vaccaro B. Crane B.R. Mechanism-based tuning of a LOV domain photoreceptor.Nat. Chem. Biol. 2009; 5: 827-834Crossref PubMed Scopus (201) Google Scholar, 43Zayner J.P. Sosnick T.R. Factors that control the chemistry of the LOV domain photocycle.PLoS One. 2014; 9e87074Crossref PubMed Scopus (73) Google Scholar, 44Hart J.E. Sullivan S. Hermanowicz P. Petersen J. Diaz-Ramos L.A. Hoey D.J. Łabuz J. Christie J.M. Engineering the phototropin photocycle improves photoreceptor performance and plant biomass production.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 12550-12557Crossref PubMed Scopus (24) Google Scholar) mutagenesis to tune various features for efficient signaling and on/off kinetics in target systems. Mechanistically, slowing the photocycle to prolong the signaling state generally involves either sterically stabilizing the photoadduct or changing the electronic state around the flavin to favor activation (26Zoltowski B.D. Vaccaro B. Crane B.R. Mechanism-based tuning of a LOV domain photoreceptor.Nat. Chem. Biol. 2009; 5: 827-834Crossref PubMed Scopus (201) Google Scholar). While specific mutations are beyond the scope of this review, we highlight the interested reader to studies that have used structure-guided mutagenesis to tune sensitivity in both optogenetic tools (45Strickland D. Lin Y. Wagner E. Hope C.M. Zayner J. Antoniou C. Sosnick T.R. Weiss E.L. Glotzer M. TULIPs: Tunable, light-controlled interacting protein tags for cell biology.Nat. Methods. 2012; 9: 379-384Crossref PubMed Scopus (350) Google Scholar, 46Kawano F. Suzuki H. Furuya A. Sato M. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins.Nat. Commun. 2015; 6: 1-8Crossref Scopus (237) Google Scholar) and plants (44Hart J.E. Sullivan S. Hermanowicz P. Petersen J. Diaz-Ramos L.A. Hoey D.J. Łabuz J. Christie J.M. Engineering the phototropin photocycle improves photoreceptor performance and plant biomass production.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 12550-12557Crossref PubMed Scopus (24) Google Scholar). In Arabidopsis, introducing mutations to tune the phot1 photocycle appeared to increase light sensitivity and plant growth under dim light conditions. However, one of the tested mutations (AtLOV2 V478L) produced a phot1 variant that exhibited autophosphorylation activity in vivo but appeared to be unable to propagate the signal downstream of light activation, as its phenotype in transgenic plants mimicked a phot1phot2 double mutant for most responses, including leaf flattening and phototropism (44Hart J.E. Sullivan S. Hermanowicz P. Petersen J. Diaz-Ramos L.A. Hoey D.J. Łabuz J. Christie J.M. Engineering the phototropin photocycle improves photoreceptor performance and plant biomass production.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 12550-12557Crossref PubMed Scopus (24) Google Scholar). This result, and others in the broader LOV signaling field, underscores the need to evaluate "tuning" mutations by a mix of assays assessing photocycle, structural, and functional properties—ideally in full-length proteins in both in vitro and cellular contexts to ensure that mutations introduce only the anticipated changes. While many LOV-containing proteins contain only a single LOV domain (31Glantz S.T. Carpenter E.J. Melkonian M. Gardner K.H.
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