Research Interests: The Lagarias Laboratory
Project Abstracts
Project 1. Molecular Mechanisms of Phytochrome Signaling. National Institutes of Health (2RO1 GM068552) J. Clark Lagarias, PI, 08-01-03 to 07-31-11
Abstract. Phytochromes are biliprotein photosensors that coordinate gene expression, growth and development of plants to optimize light harvesting when light is limiting, and to minimize light damage when light is too intense. The widespread occurrence of phytochromes in eubacteria and fungi demonstrates that these sensors are also important for heterotrophic organisms for adaptation to the circadian light environment. These studies seek to define how metabolic and light signals are perceived by phytochromes and transduced to target molecules. Our investigations address the hypothesis that bilin photoisomerization induces a 'counterion-switch' within the photoreceptor which alters ATP-dependent protein-protein interactions and phosphotransferase activities for both prokaryotic (cyanobacterial) and eukaryotic (plant) phytochrome models. These studies will use a combination of computational approaches, i.e. quantum calculations, homology modeling and molecular dynamics simulations, chemi-enzymatic synthesis of linear chromophore analogs, biochemistry and molecular biology for mutagenesis and isolation of photoreceptors with various chromophores, fluorescence, calorimetry and single-molecule assays to probe ATP- and light-modulated protein-protein interactions, together with spectroscopic analysis of wild type and phytochrome mutants in vitro. Companion experiments exploit our discovery of a fluorescent, constitutively activated allele for in vivo dissection of phytochrome signaling using a combination of fluorescence microscopy techniques and suppressor mutagenesis in the genetic model Arabidopsis thaliana. These studies are designed to test the role of ATP-binding and phosphotransferase activities in phytochrome signaling. Phytochromes are important regulatory targets, not only for minimizing plant crop yield losses to far-red enriched shade and reflected light, but also for design of drugs that target both pathogenic and beneficial microorganisms. An understanding of the molecular mechanisms of phytochrome signaling in plants is of particular significance to the developing world where inadequate crop yields and opportunistic diseases accompanying malnutrition are responsible for significant human mortality. While mammals lack this family of light sensors, phytochrome studies have already provided valuable insight into common mechanisms of cell signaling important to cancer and diabetes.
Project 2. Molecular and Structural Biology of Phycocyanobilin:Ferredoxin Oxidoreductases. National Science Foundation (PCM 0843625), Andrew J. Fisher and J. Clark Lagarias, Co-PIs, May 2009-April 2013.
Abstract. Phytobilins are heme-derived linear tetrapyrroles that perform light sensing roles in oxygenic photosynthetic organisms including green plants, cyanobacteria, and red algae. When attached to proteins (biliproteins), phytobilins function as both light detectors and light-harvesting antennae. Since biliproteins are necessary for adaptation to a changing light environment, phytobilin synthesis is critical for survival of these organisms. This proposal focuses on phycocyanobilin:ferredoxin oxidoreductase (PcyA), a representative of the ferredoxin-dependent bilin reductase (FDBR) family of catalysts responsible for formation of phycocyanobilin - the direct precursor of the chromophores of the photoreceptor phytochrome and the phycobiliprotein photosynthetic antennae. FDBRs are also found in organisms lacking both phycobiliproteins and phytochromes, including marine prochlorophytes and the primitive green alga Chlamydomonas that retains features of both plants and animals. FDBRs may therefore perform other biological roles in all organisms where they occur. Our understanding of the catalytic mechanism of these enzymes is still in its infancy since FDBRs are typically present in low abundance. Such knowledge is needed for development of activators/inhibitors of FDBRs that potentially can be used to optimize/antagonize light perception, growth and development of oxygenic photosynthetic organisms upon which all life on earth depends. For this reason, a major part of this proposal focuses on the structure and mechanism of representative cyanobacterial PcyAs using the tools of biochemistry, EPR/optical spectroscopy and X-ray crystallography. New insight into the biological function of the recently discovered PcyA in Chlamydomonas reinhardtii is the goal of a parallel line of investigation. The ability to leverage molecular tools, genetic and genomic resources available for this model photosynthetic eukaryote will enable studies to address two hypotheses regarding CrPcyA’s potential biological role(s), i.e. that CrPcyA functions to produce phytobilin precursors/activators of novel bilin/light signaling pathways and/or to regulate heme homeostasis. These investigations will not only provide insight into the structure and function of PcyA – information that is expected to facilitate design of new agonists and antagonists of the FDBR family of catalysts, but ultimately are anticipated to reveal novel bilin-dependent regulatory pathways that may also be present in animals. The broader impact of this proposal will integrate research and education by training scientists at all levels; postdocs, graduate, undergraduate and high school students, in the methods of molecular biology (cloning, site-directed mutagenesis, expression and purification of protein), enzymology (steady-state and single-turnover kinetics), protein chemistry (X-ray crystallography, chemical modification, EPR spectroscopy), computational biology (molecular modeling) and reverse genetics (Chlamydomonas biology).
Project 3. Phytochrome Engineering: A Versatile Class of Red and Near Infrared Fluorescent Protein Probes, National Science Foundation Center for Biophotonics Science and Technology (PHY-0120999), Aug 2002-July 2012.
Abstract. Phytochromes are red/near infrared switchable biliproteins that can be produced in living cells. Directed evolution of cyanobacterial phytochrome 1 (Cph1), a non-fluorescent red/far-red light absorbing photoreceptor, has been successfully used to identify an intensely red fluorescent mutant Phytofluor Red 1 – documenting the glowing potential of a new class of phytochrome-derived fluorophore for intracellular analysis. Phytochrome-based fluorescent protein probes offer several unique advantages to other fluorescent reporters: they are genetically encoded; they can readily incorporate a variety of chromophores (both natural and synthetic); and they can produce red/far-red fluorescence upon a wide range of excitation wavelengths, providing a novel family of fluorescent reporters in a previously unavailable region(s) of the spectrum. Ongoing research seeks to to characterize the biophysical properties of phytofluors (and subsequent generations of chemically evolved phytofluors), to engineer new phytofluor variants by directed evolution and to construct and evaluate phytofluor-target protein fusions for cellular applications in live cells.