The ability to sense and respond to complex environmental signals such as light, oxygen and nutrients is critical for survival and adaptation of living organisms. Many signaling proteins adopt multi-domain modular architecture to accomplish the perception of input signals and the generation of an output biological response within the same protein molecule. One example of widespread modular systems is offered by bilin-based photoreceptors in the phytochrome superfamily. Since light can readily penetrate the cell membrane, these soluble multi-domain photoreceptors offer excellent model systems for studying the still elusive mechanism of long-range signaling and allosteric regulation in modular signaling proteins such as chemoreceptors and mechanoreceptors. Our long-term goal is to understand how modular photoreceptors perceive, integrate, and transduce signals at the molecular level. To attack this goal, we adopt an integrated approach of biochemistry, spectroscopy, crystallography and cryoEM single particle reconstruction, with a main thrust on dynamic crystallography, which enables direct observation of structural responses at atomic resolution. In this proposal, we use two dual-sensor photoreceptors to represent two major types of bilin-binding photoreceptors. Both are sensory histidine kinases that feature different color perception and distinct signaling logic in response to light or chemical signals. Previously, we have obtained abundant structural information on various isolated domains by both static and dynamic crystallography. In this proposal, we will investigate the molecular mechanisms of signal integration and allosteric activation in full-length proteins where the sensor and effector domains are coupled. Specifically, we will capture structural changes in each sensory site by introducing perturbations via ligand soaking and light illumination. We will examine how the structural signals are initiated and how they propagate through the protein framework. We will jointly analyze the structures determined in different signaling states to dissect subtle motions that may involve bending, torque, winding/unwinding, or longitudinal sliding of helices. We will also perform mutagenesis and kinase assays to identify the key structural elements responsible for signal coupling between the sensor domains, the helical spine and the effector domain. We will determine the structures and dynamics of full-length photoreceptors by complementary approaches of crystallography and electron microscopy to address whether the structural asymmetry of the sensor and effector domains tethered in the same dimer scaffold plays an important role in allosteric regulation of modular photoreceptors. Our results will not only apply to photoreceptors but will inform the more general principles by which multi-domain signaling proteins such as the more widely studied chemoreceptors detect and process complex environmental signals at the molecular level. Use of light as operands in arithmetic and logic operations that override, negate, or modulate a desired cellular response is of great importance both for basic science and potentially for biotechnology applications.

Public Health Relevance

The abilities to sense and respond to complex environmental signals such as light, oxygen and nutrients are essential for survival and adaptation of living organisms. Modular photoreceptors are able to perceive and integrate multiple input signals to generate a coherent biological response. We apply biophysical methods to study the structures and dynamics of multi-sensor photoreceptors to understand how signal perception, integration and molecular logic are achieved at the molecular level. This work will provide a structural framework for repurposing and engineering of photoreceptors for biomedical research and therapeutic applications.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY024363-06
Application #
9942438
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Greenwell, Thomas
Project Start
2014-05-01
Project End
2022-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
6
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
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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