LOV to BLUF: Flavoprotein Contributions to the Optogenetic Toolkit John M. Christie, Jayde Gawthorne, Gillian Young, Niall J. Fraser, Andrew J. Roe  Molecular Plant  Volume 5, Issue 3, Pages 533-544 (May 2012) DOI: 10.1093/mp/sss020 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Optogenetic Applications Derived from Plant, Algal, and Microbial Photoreceptors. (A) Utility of ChR and NpHR expression in neurons to regulate light-driven membrane depolarization and hyperpolarization, respectively. (B) LOV2-Jα photoregulation of Rac1 activity. In darkness, LOV2 obstructs the active site of Rac1 and inhibits its activity. Blue light alleviates this steric inhibition or caging to allow Rac1 to interact with is effector protein partner p21-activated kinase 1 (PAK1). (C) Optogenetic dimerization of target proteins A and B by fusion to GI and FKF1 interaction sites, respectively, such that light instigates membrane recruitment of protein B. (D) LOV2-Jα photoregulation of stromal interaction molecule 1 (STIM1). Photoactivatable STIM1 translocates to the plasma membrane calcium channel Orai1 to generate local calcium signals upon photostimulation. Molecular Plant 2012 5, 533-544DOI: (10.1093/mp/sss020) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Domain Structures of LOV- and BLUF-Containing Photoreceptors. LOV, light, oxygen, voltage; Ser/ThrK, serine/threonine kinase; STAS, sulphate transporter antisigma-factor antagonist; HTH, helix-turn-helix; bZIP, basic region/leucine zipper; BLUF, Blue-Light-Utilizing FAD; SCHICH, sensor containing heme instead of cobalamin; EAL, cGMP phosphodiesterase motif glutamate–alanine–leucine domain; cyclase, adenylyl cyclase. Molecular Plant 2012 5, 533-544DOI: (10.1093/mp/sss020) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Structure and Photochemistry of LOV and BLUF Domains. (A) LOV2 domain of Avena sativa (oat) phot1 (PDB entry 2V0U) (Halavaty and Moffat, 2007). The structure is presented as ribbons (green) and the flavin chromophore as a stick model (blue). Position of the Jα-helix is indicated. (B) Photochemistry of the LOV domain. Light drives the formation of a thiol adduct between a conserved cysteine residue and the C4a position of the FMN chromophore. (C) BLUF domain of Klebsiella pneumoniae BlrP1 (PDB entry 2KB2) (Wu and Gardner, 2009). The structure is presented as ribbons (green) and the flavin chromophore as a stick model (blue). The Ccap region is also indicated. (D) Photochemistry of the BLUF domain. Light induces a rearrangement within the hydrogen bond network between the N5 and O4 positions of the FAD and nearby conserved tyrosine and glutamine residues. Molecular Plant 2012 5, 533-544DOI: (10.1093/mp/sss020) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Applications of LOV Fluorescence. (A) Tobacco mosaic virus (TMV)-based expression of free iLOV driven from a duplicated viral subgenomic promoter. Upper leaves of tobacco at 4 d post inoculation show systemic movement of TMV expressing the iLOV reporter (Chapman et al., 2008). (B) Monitoring Tir effector protein expression in E. coli using iLOV. Expression of Tir-iLOV can be monitored in E. coli O157:H7 (green) binding to an Embryonic Bovine Lung (EBL) cell (outlined by the dashed line). E. coli O157:H7 condenses host cell actin (blue) via its Type III secretion system (J. Gawthorne, J.M. Christie, A.J. Roe, unpublished data). This system allows extended monitoring of gene expression during host cell interactions. (C) Increasing the thermotolerance of LOV fluorescence. LOV fluorescence can be monitored in E. coli aliquots pelleted in eppendorf tubes. iLOV fluorescence is stable at 37°C, but not when cells are treated at 60°C for 10 min. In contrast, the thermostable variant tLOV is stable at both temperatures (J.M. Christie, unpublished data). (D) Production of human sterol isomerase (SI) fused to iLOV in Pichia pastoris fermenter culture. Yeast cells were grown on minimal media containing glycerol for a period of 30 h. SI production was initiated by changing the carbon source to methanol. Samples were removed throughout the fermentation, and SI production determined by measuring iLOV fluorescence per gram of wet cell weight (G. Young and N.J. Fraser, unpublished data). Molecular Plant 2012 5, 533-544DOI: (10.1093/mp/sss020) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions