Advertisement

A 1H NMR Assay for Measuring the Photostationary States of Photoswitchable Ligands

  • Matthew R. Banghart
  • Dirk Trauner
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 995)

Abstract

Incorporation of photoisomerizable chromophores into small molecule ligands represents a general approach for reversibly controlling protein function with light. Illumination at different wavelengths produces photostationary states (PSSs) consisting of different ratios of photoisomers. Thus optimal implementation of photoswitchable ligands requires knowledge of their wavelength sensitivity. Using an azobenzene-based ion channel blocker as an example, this protocol describes a 1H NMR assay that can be used to precisely determine the isomeric content of photostationary states (PSSs) as a function of illumination wavelength. Samples of the photoswitchable ligand are dissolved in deuterated water and analyzed by UV/VIS spectroscopy to identify the range of illumination wavelengths that produce PSSs. The PSSs produced by these wavelengths are quantified using 1H NMR spectroscopy under continuous irradiation through a monochromator-coupled fiber-optic cable. Because aromatic protons of azobenzene trans and cis isomers exhibit sufficiently different chemical shifts, their relative abundances at each PSS can be readily determined by peak integration. Constant illumination during spectrum acquisition is essential to accurately determine PSSs from molecules that thermally relax on the timescale of minutes or faster. This general protocol can be readily applied to any photoswitch that exhibits distinct 1H NMR signals in each photoisomeric state.

Key words

Azobenzene Photoswitch Photoisomerization Photostationary state NMR 

Notes

Acknowledgments

The authors would like to thank Jessica Harvey for contributing to the synthesis of MAQ, Rudi Nunlist of the UC Berkeley Department of Chemistry NMR facility for assisting with the NMR experiments, and Enrique Chang, formerly at Till Photonics for coordinating customization of the Polychrome V used in these experiments.

References

  1. 1.
    Ellis-Davies GC (2007) Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods 4(8):619–628PubMedCrossRefGoogle Scholar
  2. 2.
    Szobota S, Isacoff EY (2010) Optical control of neuronal activity. Annu Rev Biophys 39:329–348PubMedCrossRefGoogle Scholar
  3. 3.
    Miesenbock G (2011) Optogenetic control of cells and circuits. Annu Rev Cell Dev Biol 27:731–758PubMedCrossRefGoogle Scholar
  4. 4.
    Fehrentz T et al (2011) Optochemical genetics. Angew Chem Int Ed Engl 50(51):12156–12182PubMedCrossRefGoogle Scholar
  5. 5.
    Gorostiza P, Isacoff EY (2008) Optical switches for remote and noninvasive control of cell signaling. Science 322(5900):395–399PubMedCrossRefGoogle Scholar
  6. 6.
    Beharry AA, Woolley GA (2011) Azobenzene photoswitches for biomolecules. Chem Soc Rev 40(8):4422–4437PubMedCrossRefGoogle Scholar
  7. 7.
    Banghart MR et al (2006) Engineering light-gated ion channels. Biochemistry 45(51):15129–15141PubMedCrossRefGoogle Scholar
  8. 8.
    Rau H (2003) Azo compounds. In: Durr H, Bouas-Laurent H (eds) Photochromism: molecules and systems, revised edition. Elsevier, San Diego, pp 165–192Google Scholar
  9. 9.
    Knoll H (2004) Photoisomerism of azobenzenes. In: Horspool W, Lenci F (eds) CRC handbook of organic photochemistry and photobiology, 2nd edn. CRC Press, Boca Raton, pp 89/1–89/16Google Scholar
  10. 10.
    Hartley GS (1938) Cis form of azobenzene and the velocity of the thermal cis/trans conversion of azobenzene and some derivatives. J Chem Soc 633–642Google Scholar
  11. 11.
    LeFevre RJW, Northcott J (1953) The effects of substituents and solvents on the cis  →  trans change of azobenzene. J Chem Soc 867–870Google Scholar
  12. 12.
    Tait KM et al (2003) The novel use of NMR spectroscopy with in situ laser irradiation to study azo photoisomerization. J Photochem Photobiol A Chem 154:179–188CrossRefGoogle Scholar
  13. 13.
    Banghart M et al (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7(12):1381–1386PubMedCrossRefGoogle Scholar
  14. 14.
    Fortin DL et al (2011) Optogenetic photochemical control of designer K+  channels in mammalian neurons. J Neurophysiol 106(1):488–496PubMedCrossRefGoogle Scholar
  15. 15.
    Fischer E et al (1955) Wave length dependence of photoisomerization equilibria in azo compounds. J Chem Phys 23:1367CrossRefGoogle Scholar
  16. 16.
    Zimmerman G et al (1958) The photochemical isomerization of azobenzene. J Am Chem Soc 80:3528–3531CrossRefGoogle Scholar
  17. 17.
    Borisenko V, Woolley GA (2005) Reversibility of conformational switching in light-sensitive peptides. J Photochem Photobiol A Chem 173(1):21–28CrossRefGoogle Scholar
  18. 18.
    Banghart MR et al (2009) Photochromic blockers of voltage-gated potassium channels. Angew Chem Int Ed Engl 48(48):9097–9101PubMedCrossRefGoogle Scholar
  19. 19.
    Blaustein RO et al (2000) Tethered blockers as molecular ‘tape measures’ for a voltage-gated K+  channel. Nat Struct Biol 7(4):309–311PubMedCrossRefGoogle Scholar
  20. 20.
    Blaustein RO (2002) Kinetics of tethering quaternary ammonium compounds to K(+) channels. J Gen Physiol 120(2):203–216PubMedCrossRefGoogle Scholar
  21. 21.
    Sawicki E (1957) Physical properties of aminoazobenzene dyes. VIII. Absorption spectra in acid solution. J Org Chem 22:1084–1088CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Matthew R. Banghart
    • 1
  • Dirk Trauner
    • 2
    • 3
  1. 1.Department of NeurobiologyHarvard Medical SchoolBostonUSA
  2. 2.Department of ChemistryLudwig-Maximillians-Universität-University of MunichMunichGermany
  3. 3.Center for Integrated Protein ScienceLudwig-Maximillians-Universität-University of MunichMunichGermany

Personalised recommendations