Photochemistry of flavoprotein light sensors

Conrad, Karen S.; Manahan, C.C.; Crane, Brian R.

doi:10.1038/nchembio.1633

Cited by 205 publications

(243 citation statements)

References 110 publications

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“…The simplest proposed sensing mechanism is a rotamer shift of the glutamine, so that after stimulation, this side chain donates a hydrogen bond to the C4=O carbonyl and accepts one from the tyrosine, whereas in the dark state, the glutamine donates to the tyrosine (11,13,14). In BlrP1, these changes are accompanied by movement of a nearby methionine on the β5-strand of the BLUF domain, and both "Met in " and "Met out " arrangements have been described (15).…”

Section: Resultsmentioning

confidence: 99%

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Ohki

Sugiyama

Kawai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.optogenetics | X-ray crystallography | blue light | allostery

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Section: Resultsmentioning

confidence: 99%

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Ohki

Sugiyama

Kawai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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Section: Introductionmentioning

confidence: 99%

“…Similar magnetosensitivity is conceivable in other cryptochrome-derived radical pairs in which FAD  or its protonated form, FADH  , is paired with an ascorbic acid radical 14 or, less plausibly, superoxide, 2 O ·- 15,16 . There appear to be different electron transfer pathways in some cryptochromes 17,18 , but no evidence so far that they give rise to magnetic field effects.The radical pair mechanism is well established as the source of magnetic field effects on chemical reactions 19 . Remarkably, the magnetic interactions of electron spins in organic radicals can result in significant changes in reaction kinetics and product yields even though those interactions are many orders of magnitude weaker than the thermal energy, k B T. The sensitivity to applied magnetic fields derives from the coherent spin dynamics of pairs of radicals formed in spincorrelated states.…”

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confidence: 99%

Chemical amplification of magnetic field effects relevant to avian magnetoreception

et al. 2016

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Magnetic fields as weak as the Earth's can change the yields of radical pair reactions even though the energies involved are orders of magnitude smaller than k B T at room temperature. Proposed as the source of the light-dependent magnetic compass in migratory birds, the radical pair mechanism is thought to operate in cryptochrome flavoproteins in the retina. Here we demonstrate that the primary magnetic field effect on flavin photoreactions can be chemically amplified by slow radical termination reactions under conditions of continuous photoexcitation. The nature and origin of the amplification are revealed by studies of the intermolecular flavin-tryptophan and flavin-ascorbic acid photocycles and the closely related intramolecular flavin-tryptophan radical pair in cryptochrome. Amplification factors of up to 5.6 have been observed for magnetic fields weaker than 1 mT. Substantial chemical amplification could have a significant impact on the viability of a cryptochrome-based magnetic compass sensor.2 Amongst other directional cues, migratory birds use a light-dependent geomagnetic compass for orientation and navigation [1][2][3] . Although the primary detection mechanism is unclear, the evidence currently points to magnetically sensitive photochemical reactions in cryptochrome proteins located in the retina [4][5][6] . Cryptochromes 7 contain the chromophore flavin adenine dinucleotide (FAD), photoexcitation of which can trigger three consecutive intra-protein electron transfers along a conserved triad of tryptophan (Trp) residues to produce a (FAD  Trp  ) radical pair [8][9][10] . This form of cryptochrome is magnetically sensitive in vitro and possibly also in vivo [11][12][13] . Similar magnetosensitivity is conceivable in other cryptochrome-derived radical pairs in which FAD  or its protonated form, FADH  , is paired with an ascorbic acid radical 14 or, less plausibly, superoxide, 2 O ·- 15,16 . There appear to be different electron transfer pathways in some cryptochromes 17,18 , but no evidence so far that they give rise to magnetic field effects.The radical pair mechanism is well established as the source of magnetic field effects on chemical reactions 19 . Remarkably, the magnetic interactions of electron spins in organic radicals can result in significant changes in reaction kinetics and product yields even though those interactions are many orders of magnitude weaker than the thermal energy, k B T. The sensitivity to applied magnetic fields derives from the coherent spin dynamics of pairs of radicals formed in spincorrelated states. Several conditions need to be satisfied for a radical pair to be suitable as a geomagnetic compass sensor 5 : (a) the electron spin in at least one of the radicals must interact anisotropically with nuclear spins; (b) the mutual interaction of the two electron spins should be small; (c) the radical pair must react spin-selectively; (d) its lifetime should not be too short; and (e) the electron spin relaxation must be relatively slow. Studies of isolated cry...

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“…light sensing | flavoprotein | photochemistry | redox | molecular dynamics C ryptochromes (CRYs) are flavin-binding proteins that perform a variety of sensory and catalytic functions in all kingdoms of life (1,2). CRYs are closely related to the DNA photolyases (PLs), which catalyze light-driven redox reactions to break apart pyrimidine dimers in UV-damaged DNA (1,2).…”

mentioning

confidence: 99%

Changes in active site histidine hydrogen bonding trigger cryptochrome activation

Ganguly

Manahan

Top

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pK a . Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.light sensing | flavoprotein | photochemistry | redox | molecular dynamics C ryptochromes (CRYs) are flavin-binding proteins that perform a variety of sensory and catalytic functions in all kingdoms of life (1, 2). CRYs are closely related to the DNA photolyases (PLs), which catalyze light-driven redox reactions to break apart pyrimidine dimers in UV-damaged DNA (1, 2). CRYs and PLs share a conserved photolyase homology region that consists of an α-helical domain, which binds flavin adenine dinucleotide (FAD) and an α/β Rossman-fold domain, which sometimes binds a pteridine or deazaflavin antenna cofactor. CRYs also contain C-terminal extensions of variable sizes that contribute to their specific functions. The range of activities found for CRYs and PLs require that their flavin cofactors assume a broad range of redox, protonation, and excited states (1, 2).In the fruit fly Drosophila melanogaster, a type I cryptochrome (dCRY) is the primary light receptor of the circadian clock (1, 3). In response to blue light, dCRY coordinates interactions between Timeless (TIM) and the E3-ubiquitin ligase Jetlag (JET) (4). JETmediated proteolysis of TIM destabilizes its partner Period (PER). PER serves as the principal repressor of circadian gene expression and its degradation phase-shifts the clock (3). dCRY also catalyzes light-induced self-degradation that involves another E3-ligase: Brwd3 or RAMSHACKLE (5)...

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Photochemistry of flavoprotein light sensors

Cited by 205 publications

References 110 publications

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Chemical amplification of magnetic field effects relevant to avian magnetoreception

Changes in active site histidine hydrogen bonding trigger cryptochrome activation

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