Among the environmental factors that influence biology, light controls an incredibly rich set of processes including circadian rhythms, movement and development. Central to this regulation are diverse families of photoreceptors that harness the photochemically-triggered configurational changes of light-sensitive cofactors, the chemical properties of which confer sensitivity to specific portions of the UV and visible spectrum. These structural alterations are amplified as allosteric changes in the surrounding protein, initiating signal transduction pathways that control biological responses. Understanding how these initial sensing and signaling events are triggered has given insight into fundamental aspects of biology and enabled the development of novel optogenetic tools that are enabling discoveries in diverse fields of biomedicine. Here we focus on examining both signaling mechanism and applicability to tool development for proteins containing LOV (Light-Oxygen- Voltage) photosensory domains. These domains are found in thousands of proteins to date, controlling the activity of over 20 enzymatic and non-enzymatic effector domains in natural systems and many others in engineered proteins. To do so, LOV domains undergo the specific photochemical formation of protein/flavin adducts upon illumination with blue light, using this as a trigger to allosterically drive protein conformational changes around the flavin. This configurational change is allosterically transmitted to the surrounding protein, providing a light-based ?switch? of protein activity that remains engaged until illumination ceases. Fundamental questions regarding these signaling processes remain unanswered, limiting our understanding of natural and engineered LOV proteins. We propose to answer these limitations by pursuing three aims: 1). Determine the lit state structures of two classes of activated LOV proteins to give insight into signaling and guiding future engineering efforts; 2). Establish the correlation of in vitro photochemical and DNA-binding parameters on cellular function of a LOV-controlled transcription factor tools; 3). Examine the generality of LOV signaling models by determining the structural role of photosensing in a novel class of RGS-LOV signaling proteins. To achieve these ends, we will take advantage of an broad foundation of preliminary structural and functional data that will be extended with a mix of biophysical and biochemical studies. Outcomes from this research will include information about fundamental regulatory processes employed by these proteins, giving insights that will be broadly applicable for signal transduction studies.

Public Health Relevance

The proposed research will give valuable information into the mechanisms used by LOV proteins, a widespread group of photosensory proteins activated by blue light, to sense and respond to changes in their environment. Such insight is fundamental to understanding the basis of photobiological responses throughout nature, ranging from the entrainment of circadian rhythms to the infectivity of pathogenic bacteria. Further, this mechanistic information is critical to the development of novel ?optogenetic? light-regulated protein reagents, giving biomedical researchers new ways to control cellular function with unprecedented precision.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM106239-08S1
Application #
9989318
Study Section
Program Officer
Nie, Zhongzhen
Project Start
2013-09-30
Project End
2021-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
8
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Advanced Science Research Center
Department
Type
Organized Research Units
DUNS #
831361857
City
New York
State
NY
Country
United States
Zip Code
10031
Glantz, Spencer T; Berlew, Erin E; Jaber, Zaynab et al. (2018) Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids. Proc Natl Acad Sci U S A 115:E7720-E7727
Reade, Anna; Motta-Mena, Laura B; Gardner, Kevin H et al. (2017) TAEL: a zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control. Development 144:345-355
Clark, Lindsay D; Dikiy, Igor; Chapman, Karen et al. (2017) Ligand modulation of sidechain dynamics in a wild-type human GPCR. Elife 6:
Glantz, Spencer T; Carpenter, Eric J; Melkonian, Michael et al. (2016) Functional and topological diversity of LOV domain photoreceptors. Proc Natl Acad Sci U S A 113:E1442-51
Clark, Lindsay; Zahm, Jacob A; Ali, Rustam et al. (2016) Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris. J Biomol NMR 64:267
CorrĂȘa, Fernando; Gardner, Kevin H (2016) Basis of Mutual Domain Inhibition in a Bacterial Response Regulator. Cell Chem Biol 23:945-954
Ocasio, Victor J; CorrĂȘa, Fernando; Gardner, Kevin H (2015) Ligand-induced folding of a two-component signaling receiver domain. Biochemistry 54:1353-63
Clark, Lindsay; Zahm, Jacob A; Ali, Rustam et al. (2015) Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris. J Biomol NMR 62:239-45
Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R et al. (2014) Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation. Proc Natl Acad Sci U S A 111:17839-44
Motta-Mena, Laura B; Reade, Anna; Mallory, Michael J et al. (2014) An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol 10:196-202