Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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 α-helix (Jα) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain–Jα-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by 3.8 kcal mol−1, thus quantifying the energy available through LOV-Jα 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-Jα interaction.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Schematic representations of phototropin domain organization and photoswitching mechanism.
Figure 2: Conformational exchange dynamics at the LOV-Jα interface as detected by relaxation dispersion measurements.
Figure 3: 15N relaxation dispersion analyses of LOV2-Jα constructs containing V529A, V529E and V529 point mutations.
Figure 4: The Jα-helix is unfolded in the high-energy conformation of dark state LOV2-Jα.
Figure 5: The Jα-helix is largely unfolded in the lit state.


  1. 1

    Bhattacharyya, R.P., Remenyi, A., Yeh, B.J. & Lim, W.A. Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits. Annu. Rev. Biochem. 75, 655–680 (2006).

    CAS  Article  Google Scholar 

  2. 2

    Pawson, T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116, 191–203 (2004).

    CAS  Article  Google Scholar 

  3. 3

    van der Horst, M.A. & Hellingwerf, K.J. Photoreceptor proteins, “Star actors of modern times”: a review of the functional dynamics in the structure of representative members of six different photoreceptor families. Acc. Chem. Res. 37, 13–20 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Christie, J.M. Phototropin blue-light receptors. Annu. Rev. Plant Biol. 58, 21–45 (2007).

    CAS  Article  Google Scholar 

  5. 5

    Huala, E. et al. Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278, 2120–2123 (1997).

    CAS  Article  Google Scholar 

  6. 6

    Taylor, B.L. & Zhulin, I.B. PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev. 63, 479–506 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Crosson, S., Rajagopal, S. & Moffat, K. The LOV domain family: photoresponsive signaling modules coupled to diverse output domains. Biochemistry 42, 2–10 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Christie, J.M., Salomon, M., Nozue, K., Wada, M. & Briggs, W.R. LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc. Natl. Acad. Sci. USA 96, 8779–8783 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Crosson, S. & Moffat, K. Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch. Plant Cell 14, 1067–1075 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Christie, J.M., Swartz, T.E., Bogomolni, R.A. & Briggs, W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function. Plant J. 32, 205–219 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Fedorov, R. et al. Crystal structures and molecular mechanism of a light-induced signaling switch: The phot-LOV1 domain from Chlamydomonas reinhardtii. Biophys. J. 84, 2474–2482 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Crosson, S. & Moffat, K. Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction. Proc. Natl. Acad. Sci. USA 98, 2995–3000 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Harper, S.M., Neil, L.C. & Gardner, K.H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Zoltowski, B.D. et al. Conformational switching in the fungal light sensor vivid. Science 316, 1054–1057 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Halavaty, A.S. & Moffat, K. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry 46, 14001–14009 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Möglich, A. & Moffat, K. Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA. J. Mol. Biol. 373, 112–126 (2007).

    Article  Google Scholar 

  17. 17

    Corchnoy, S.B. et al. Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1. J. Biol. Chem. 278, 724–731 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Harper, S.M., Christie, J.M. & Gardner, K.H. Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity. Biochemistry 43, 16184–16192 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Carver, J.P. & Richards, R.E. A general two-site solution for the chemical exchange produced dependence of T2 upon Carr-Purcell pulse separation. J. Magn. Reson. 6, 89–105 (1972).

    CAS  Google Scholar 

  20. 20

    Palmer, A.G. III, Kroenke, C.D. & Loria, J.P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol. 339, 204–238 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Harper, S.M., Neil, L.C., Day, I.J., Hore, P.J. & Gardner, K.H. Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy. J. Am. Chem. Soc. 126, 3390–3391 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Genick, U.K., Soltis, S.M., Kuhn, P., Canestrelli, I.L. & Getzoff, E.D. Structure at 0.85 A resolution of an early protein photocycle intermediate. Nature 392, 206–209 (1998).

    CAS  Article  Google Scholar 

  23. 23

    van der Horst, M.A., van Stokkum, I.H., Crielaard, W. & Hellingwerf, K.J. The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation. FEBS Lett. 497, 26–30 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Harigai, M., Imamoto, Y., Kamikubo, H., Yamazaki, Y. & Kataoka, M. Role of an N-terminal loop in the secondary structural change of photoactive yellow protein. Biochemistry 42, 13893–13900 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Nolen, B., Taylor, S. & Ghosh, G. Regulation of protein kinases; controlling activity through activation segment conformation. Mol. Cell 15, 661–675 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Buchwald, G. et al. Conformational switch and role of phosphorylation in PAK activation. Mol. Cell. Biol. 21, 5179–5189 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Gardino, A.K. & Kern, D. Functional dynamics of response regulators using NMR relaxation techniques. Methods Enzymol. 423, 149–165 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Volgraf, M. et al. Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat. Chem. Biol. 2, 47–52 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Gorostiza, P. et al. Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor. Proc. Natl. Acad. Sci. USA 104, 10865–10870 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Hausser, M. & Smith, S.L. Neuroscience: controlling neural circuits with light. Nature 446, 617–619 (2007).

    Article  Google Scholar 

  31. 31

    Schroder-Lang, S. et al. Fast manipulation of cellular cAMP level by light in vivo. Nat. Methods 4, 39–42 (2007).

    Article  Google Scholar 

  32. 32

    Dueber, J.E., Yeh, B.J., Chak, K. & Lim, W.A. Reprogramming control of an allosteric signaling switch through modular recombination. Science 301, 1904–1908 (2003).

    CAS  Article  Google Scholar 

  33. 33

    Rosen, M.K. et al. Selective methyl group protonation of perdeuterated proteins. J. Mol. Biol. 263, 627–636 (1996).

    CAS  Article  Google Scholar 

  34. 34

    Lee, A.L., Urbauer, J.L. & Wand, A.J. Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins. J. Biomol. NMR 9, 437–440 (1997).

    CAS  Article  Google Scholar 

  35. 35

    Loria, J.P., Rance, M. & Palmer, A.G. III. A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J. Am. Chem. Soc. 121, 2331–2332 (1999).

    CAS  Article  Google Scholar 

  36. 36

    Skrynnikov, N.R., Mulder, F.A., Hon, B., Dahlquist, F.W. & Kay, L.E. Probing slow time scale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: application to methionine residues in a cavity mutant of T4 lysozyme. J. Am. Chem. Soc. 123, 4556–4566 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Tollinger, M., Skrynnikov, N.R., Mulder, F.A., Forman-Kay, J.D. & Kay, L.E. Slow dynamics in folded and unfolded states of an SH3 domain. J. Am. Chem. Soc. 123, 11341–11352 (2001).

    CAS  Article  Google Scholar 

  38. 38

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Article  Google Scholar 

  39. 39

    Johnson, B.A. & Blevins, R.A. NMRView - a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    CAS  Article  Google Scholar 

  40. 40

    Panchal, S.C., Bhavesh, N.S. & Hosur, R.V. Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins. J. Biomol. NMR 20, 135–147 (2001).

    CAS  Article  Google Scholar 

  41. 41

    Kay, L.E., Xu, G.Y. & Yamazaki, T. Enhanced-sensitivity triple-resonance spectroscopy with minimal H2O saturation. J. Magn. Reson. A 109, 129–133 (1994).

    CAS  Article  Google Scholar 

  42. 42

    Wittekind, M. & Mueller, L. HNCACB, a high-sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the alpha- and beta-carbon resonances in proteins. J. Magn. Reson. B 101, 201–205 (1993).

    CAS  Article  Google Scholar 

  43. 43

    Grzesiek, S. & Bax, A. Correlating backbone amide and side-chain resonances in larger proteins by multiple relayed triple resonance NMR. J. Am. Chem. Soc. 114, 6291–6293 (1992).

    CAS  Article  Google Scholar 

  44. 44

    Rubinstenn, G. et al. Structural and dynamic changes of photoactive yellow protein during its photocycle in solution. Nat. Struct. Biol. 5, 568–570 (1998).

    CAS  Article  Google Scholar 

  45. 45

    Cavanagh, J., Fairbrother, W.J., Palmer, A.G., Rance, M. & Skelton, N.J. Protein NMR Spectroscopy: Principles and Practice 1–687 (Academic Press, San Diego, 2007).

    Google Scholar 

  46. 46

    DeLano, W.L. The PyMOL Molecular Graphic System (DeLano Scientific, Palo Alto, California, USA, 2002).

    Google Scholar 

Download references


Research was supported by grants from the Welch Foundation to M.K.R. (I–1544) and K.H.G. (I–1424) and by a grant from the Texas Higher Education Coordinating Board Advanced Technology Program (010019-0124-2003). We thank L.E. Kay (University of Toronto) for providing the pulse sequences and software for data acquisition and analyses, D.M. Korzhnev (University of Toronto) for help with experimental setup and data fitting and D. Kern (Brandeis University) for assistance with improved data acquisition methods. We also thank all members of the Rosen and Gardner labs for helpful discussions.

Author information




X.Y., M.K.R. and K.H.G. designed experiments, analyzed data and wrote the paper, and X.Y. performed experiments.

Corresponding author

Correspondence to Kevin H Gardner.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 2980 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yao, X., Rosen, M. & Gardner, K. Estimation of the available free energy in a LOV2-Jα photoswitch. Nat Chem Biol 4, 491–497 (2008).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing