Estimation of the available free energy in a LOV2-Jα photoswitch

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8816, USA.
Nature Chemical Biology (Impact Factor: 13). 09/2008; 4(8):491-7. DOI: 10.1038/nchembio.99
Source: PubMed


Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins--plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent alpha-helix (J alpha) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain-J alpha-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by approximately 3.8 kcal mol(-1), thus quantifying the energy available through LOV-J alpha for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-J alpha interaction.

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Available from: Kevin H Gardner, Oct 04, 2015
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    • "Light-sensingdomainshavebeenusedtoallostericallycontrolproteinfunction .AsLOV2hasbeenused mostprominentlyforthispurpose,asthisdomainis small,modular,wellcharacterisedandundergoesa largestructuralchangeuponphotoexcitation.Generally,therehavebeentwostrategiesforphotoreg- ulationusingAsLOV2:(i)AsLOV2–Jαisattached toatargetproteinsuchthatconformationalchanges inLOV2induceconformationalchangesinthetar- getand(ii)stericocclusion–adomainisattachedto AsLOV2–Jαinsuchawaythatbindingtoaneffector isblockedinthedarkbutpermittedinthelightupon dissociationofJαfromtheLOVcore.Takingtheformerapproach ,theAsLOV2–Jαdomainwasinserted atdifferentsiteswithintheenzymedihydrofolate reductase,resultinginlight-regulateddihydrofolate reductaseactivity(Leeetal.,2008).AsLOV2–Jαhas alsobeenusedtocontrolactivityoflipaseA(Krauss etal.,2010).Inanotherstudy,AsLOV2–Jαwasat- tachedtoanN-terminalhelixofthebacterialtrp repressor,TrpR(Stricklandetal.,2008).Thefusion protein,'LovTAP',wasfoundtobindDNApreferentiallyinlight ,demonstratingthatchangesarisingin theJα-helixuponphotoexcitationweretransferred toinduceaconformationalchangeinTrpR. PriorstudiesoftheinteractionofAsLOV2and theC-terminalJα-helixhavedemonstratedthatthe Jα-helixisnotsimplyboundtotheLOVcorein thedarkandreleasedinlight,butpopulatesboth statesinlightanddark,althoughatdifferentratios (Yaoetal.,2008).Mutationsthatdestabilisedocking totheLOVcorewereidentifiedandfoundtoleadto light-independentconstitutiveactivity(Harperetal., 2004).Inafollow-uptotheLovTAPstudy(Stricklandetal .,2008),theauthorsidentifiedmutations inLOV–JαthatstabilisedLOVdocking,increasing thedynamicrangeoftheDNA-bindingphotoswitch fromafivefoldto70-folddifferenceinDNA-binding affinitybetweenlitanddarkstates(Stricklandetal., 2010). "
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    ABSTRACT: Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometer precision within cells. While light-actuated chemicals such as photolabile "caged" compounds have been in existence for decades, the use of genetically-encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.
    Biology of the Cell 11/2012; 105(2). DOI:10.1111/boc.201200056 · 3.51 Impact Factor
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    • "It was somewhat surprising that the SsrA peptide had significantly lower lit state affinity for SspB when embedded in the N-terminal portion of the Jα helix than when placed at the C-terminus. NMR experiments with the WT AsLOV2 domain indicate that the helix undocks as a cooperative unit when the protein is activated with light (Yao, et al., 2008). This suggests that there should be similar levels of access to residues in the N-terminal and C-terminal regions of the Jα helix in the lit state, and therefore, we expected that lit-state binding affinity for the peptide would be relatively insensitive to where the peptide was placed in the Jα helix. "
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    ABSTRACT: Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Jα helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49- and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light-dependent colocalization, which we demonstrate with photo-activable gene transcription in yeast.
    Chemistry & biology 04/2012; 19(4):507-17. DOI:10.1016/j.chembiol.2012.02.006 · 6.65 Impact Factor
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    • "In their seminal studies, Gardner and co-workers identified an amphipathic helix (designated Ja; Figure 3A) C-terminal to the LOV2 domain of Avena sativa (oat) phot1 that docks onto the b-scaffold of the LOV2-core in darkness (Harper et al., 2003). Photostimulation of LOV2 causes the Ja-helix to undock from the b-scaffold (Harper et al., 2003; Yao et al., 2008)—a process that correlates with an activation of the C-terminal kinase domain of phot1 (Harper et al., 2004). Displacement of Ja is driven largely by light-induced distortions propagated within the b-scaffold (Nozaki et al., 2004; Jones et al., 2007; Nash et al., 2008). "
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    ABSTRACT: Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.
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