Biological photoreceptors are exceptionally interesting objects of study, not only in relation to their biological function but also because they can be triggered by a short flash of light, allowing the study of functional protein dynamics over a wide span of timescales. In an important new development, biological photoreceptors have been engineered for use as photonic switches and sensors in cells and tissues. They have a modular architecture with conserved ‘input’ and ‘output’ domains, which can be swapped so that an effector domain with desired properties can be rendered light-sensitive. As the light-absorbing chromophore often is a naturally occurring cofactor in cells (such as flavins or biliverdin), photoreceptor proteins can readily be genetically encoded. The light activation mechanisms of biological photoreceptors are therefore of considerable significance for biomedical research and technology. Thus, biological photoreceptors represent true biological photonic material, tailored for efficient optical sensing and switching through natural selection and amenable to engineering through their modular architecture.
To understand the complex relation between photon absorption and the resulting dynamics that characterize the photoswitched and photosensory function, the structural changes upon light activation need to be determined. We are running a research program on flavin-binding photoreceptors: the LOV and BLUF domains. We aim to resolve their photochemical reaction mechanism, how this mechanism alters the local structure around the flavin cofactor, how this local structural perturbation is transmitted to the molecular surface and in what way the molecular surface is affected. To this end, we employ novel, state-of-the-art time-resolved optical and vibrational techniques to determine the functional-structural changes and their associated timescales that make LOV and BLUF photoreceptors work, and thus understand its photoswitched function at the molecular level. Importantly, we use LOV and BLUF domains with isotopically labeled flavin chromophores, as well as isotopically labeled amino acids incorporated at critical ‘hot spots’ in the protein. The unique combination of isotopically labeled flavin and amino acids, and time-resolved vibrational spectroscopy will enable unambiguous assignment of transient local structure, in space and in time. This powerful multidisciplinary approach will enable a thorough understanding of the photoswitched function of LOV and BLUF domains and the resulting insights may form a basis for rational engineering of biophotonic switches.
For more information, contact Dr. John Kennis, firstname.lastname@example.org, +31 (0)205987212.
K.C. Toh, E.A. Stojkovic, I.H.M. van Stokkum, K. Moffat, J.T.M. Kennis
Proton transfer and hydrogen bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome
Proc. Natl. Acad. Sci. (USA) 107, (2010) 9170 - 9175
C. Bonetti, T. Mathes, I.H.M. van Stokkum, K.M. Mullen, M.L. Groot, R. van Grondelle, P. Hegemann, J.T.M. Kennis
Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy
Biophys. J. 95, 2008, pp. 4790 - 4802
J.T.M. Kennis and M.L. Groot
Ultrafast spectroscopy of biological photoreceptors
Curr. Opinion Struct. Biol 17 (2007), p. 623-630
M. Gauden, I.H.M. van Stokkum, J.M. Key, D. Ch. Luhrs, R. van Grondelle, P. Hegemann, J.T.M. Kennis
Hydrogen bond switching via a radical pair mechanism in a flavin-binding photoreceptor
Proc. Natl. Acad. Sci. USA 103, 2006, p. 10895-10900
M. Gauden, S. Yeremenko, W. Laan, I.H.M. van Stokkum, J.A. Ihalainen, R. van Grondelle, K.J. Hellingwerf, J.T.M. Kennis
Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional anti-repressor of photosynthesis genes
Biochemistry 44, 2005, p. 3653-3662
J.T.M. Kennis, S. Crosson, M. Gauden, I.H.M. van Stokkum, K. Moffat, R. van Grondelle
Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor
Biochemistry 42, 2003, p. 3385-3392