Light is a fundamental environmental signal for living organisms. Photoreceptors convert light signals into biochemical and biological signals that ultimately regulate a wide range of important physiological processes such as photosynthesis, circadian rhythm and vision. Our long-term goal is to understand the signaling mechanisms of photoreceptors at the molecular level. We integrate crystallography, X-ray solution scattering, spectroscopic and biochemical approaches to investigate structures and signaling mechanisms of the red-/far-red-light photoreceptors, bacteriophytochromes (BphPs). BphPs absorb and respond to photons in the long wavelength range of the visible solar spectrum between 650nm and 900nm. A typical BphP photo-converts reversibly between red-light- absorbing (Pr) and far red-light-absorbing (Pfr) states, in which its C-terminal histidine kinase (HK) domain undergoes light-dependent auto-phosphorylation and then relays the phosphate group to a downstream response regulator in a two-component signaling pathway. At the center of this research are three core questions on signaling in BphPs. 1) What conformational changes are triggered in the chromophore upon absorbing a photon? 2) What is the nature of light-induced structural signals in the protein moiety? 3) How are local structural signals transmitted from the chromophore-binding site to the active site of the spatially distant HK? We address these questions by examining structures, dynamics and kinetic pathways of light-induced molecular events in three representative BphPs, on a wide range of time and length scales. We apply both static crystallography and mutagenesis to identify key structural elements and interactions in distinct signaling states. We conduct pump-probe experiments to initiate and follow photoreactions by X-ray scattering from both crystals and solutions of photoactive BphPs, to directly observe light-induced structural changes at room temperature. We thus explore the mechanism by which a red light signal is converted into a biological signal at the molecular level. In addition, the principles of long-range signal transduction in BphPs will have broader implications for understanding molecular mechanisms of more widespread, modular signaling proteins such as chemoreceptors. Since the range of the action spectrum of BphPs coincides with the therapeutic optical window for humans, BphPs have great potential as red fluorescent proteins for deep tissue fluorescent imaging and as genetically encoded tools for optical manipulation of in vivo functions. Our findings will serve as a structural framework to guide further development of BphP-based biomedical applications.

Public Health Relevance

The ability to perceive and respond to light signals is essential for life on earth to survive and adapt to its ever-changing environment. Photoreceptors are responsible for mediating light responses in living organisms by converting a physical light signal into a biological signal. We will apply biophysical approaches to understand how red light photoreceptors work at the molecular level. Success will also offer new strategies to develop genetically encoded optical tools for biomedical research and therapeutic applications.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Greenwell, Thomas
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University of Chicago
Schools of Medicine
United States
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Ren, Zhong; Ayhan, Medine; Bandara, Sepalika et al. (2018) Crystal-on-crystal chips for in situ serial diffraction at room temperature. Lab Chip 18:2246-2256
Bandara, Sepalika; Ren, Zhong; Lu, Lu et al. (2017) Photoactivation mechanism of a carotenoid-based photoreceptor. Proc Natl Acad Sci U S A 114:6286-6291
Ren, Zhong; Yang, Xiaojing (2016) Angular-split/temporal-delay approach to ultrafast protein dynamics at XFELs. Acta Crystallogr D Struct Biol 72:871-82
Bertoni, R; Lorenc, M; Graber, T et al. (2016) Cooperative elastic switching vs. laser heating in [Fe(phen)2(NCS)2] spin-crossover crystals excited by a laser pulse. CrystEngComm 18:7269-7275
Ren, Zhong (2016) Molecular events during translocation and proofreading extracted from 200 static structures of DNA polymerase. Nucleic Acids Res 44:7457-74
Ren, Zhong; Ren, Peter X; Balusu, Rohith et al. (2016) Transmembrane Helices Tilt, Bend, Slide, Torque, and Unwind between Functional States of Rhodopsin. Sci Rep 6:34129
Pande, Kanupriya; Hutchison, Christopher D M; Groenhof, Gerrit et al. (2016) Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352:725-9
Wang, Cheng; Flanagan, Moira L; McGillicuddy, Ryan D et al. (2016) Bacteriophytochrome Photoisomerization Proceeds Homogeneously Despite Heterogeneity in Ground State. Biophys J 111:2125-2134
Yang, Xiaojing; Stojkovi?, Emina A; Ozarowski, Wesley B et al. (2015) Light Signaling Mechanism of Two Tandem Bacteriophytochromes. Structure 23:1179-89
Yang, Xiaojing; Montano, Sherwin; Ren, Zhong (2015) How Does Photoreceptor UVR8 Perceive a UV-B Signal? Photochem Photobiol 91:993-1003

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