| Bogomolni Research Lab | ||||||||
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| Research |
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Biological Light Sensors and Light Energy Transducers Key Words: Retinal Proteins, LOV domain photoreceptor proteins, Photosensory Transduction, Membrane Bioenergetics, Photochemistry, Laser Flash Photolysis, absorption and fluorescence spectroscopy, vibration spectroscopy, Electron Paramagnetic Resonance, Nuclear Magnetic Resonance, Mass Spectrometry, bacterial phototaxis, bacterial virulence. My research focuses on the mechanisms of light energy conversion and light signal transduction in biological systems, and their evolution. Light is the primary source of energy in the biosphere as well as the stimulus that provides environmental information to living organisms. We aim to elucidate the molecular structures involved and the molecular mechanism that these systems use to convert light energy into energy forms suitable to drive life processes, or to sense light and transfer the information to the cell to elicit a physiological response. Bacterial Rhodopsins In the archaebacterium halobacterium salinarum, both light energy conversion and light signal transduction are carried out by a family of intrinsic membrane proteins that contain retinal chromophores. Two of these rhodopsinlike proteins, bacteriorhodopsin (bR) and halorhodopsin (hR), are light-driven ion pumps (for protons and chloride, respectively). They harvest light energy and store it as a transmembrane electrochemical potential. In addition, the halobacterium's motility is modulated by light, which can have an attractant or repellent effect, depending on wavelength. This primitive color-sensing mechanism enables the cell to migrate into an environment optimal for light absorption by bR and hR. The sensory photoreceptors are two additional retinal pigments, the bacterial sensory rhodopsins I and II (sR-I and sR-II), which are chemically similar to bR and hR but do not function as electrogenic ion pumps. Structurally these proteins consist of seven transmembrane helices connected at the cell surfaces by inter-helical loops and contain covalently bound retinal (a vitamin A relative) as the light sensitive element. Upon light activation both the ion pumping bacterial rhodopsins as well as the sensory rhodopsins undergo a series of retinal and protein conformation changes that elicit outward proton translocation across the membrane in Bacteriorhodopsin or inward chloride translocation in Halorhodopsin, effectively converting light energy into electrochemical potential energy. The bacterial sensory rhodopsins are not ion pumps. Instead, protein conformation changes propagate to associated specific transducer proteins that relay the signal to the cell. LOV-Domain Blue Light Photoreceptors For the past 12 years we have focused on a new family of photoreceptors, the LOV-domain proteins (LOV stands for, Light-Oxygen-Voltage). The LOV domains are small photoactive ca. 10 kD protein modules that contain a non-covalently bound flavin (vitamin B2 related) as their light sensitive element. A LOV protein contains in addition to its LOV module one or more output domains that carry out a biological function activated by blue light absorbed by the LOV domain. Typically the LOV proteins are not integral membrane proteins but can be membrane associated. The first members of this family were the plant phototropins, which are the light sensors for several responses of plants to blue light, such as phototropism, stomata opening, and chloroplast relocation in cells. LOV domains undergo a light reaction that results in the formation of a covalent adduct between the flavin and a universally conserved reactive cysteine residue. This reaction causes a local structural perturbation that propagates to the rest of the protein and activates biochemical functions of the output domains. Over 100 different LOV domain proteins have been identified in the genomes of all three life domains: eubacteria, archaea, and eukarya. Based on their genome sequences the different output domains are predicted to have a variety of functions which include among them various types of enzymatic activities, DNA binding and transcription factor activation and gene expression regulation. We have recently discovered that some of these flavin photoreceptors have a role in blue light activation of virulence in animal and human pathogenic bacteria and on the establishment of symbiotic interaction between nitrogen fixing rhizobial bacteria and leguminous plants. Our current research aims at understanding the molecular mechanisms of activation of the LOV domains, and the elucidation of the structures and mechanisms involved in intramolecular signal relay, and on the cellular signal transduction pathways. The experimental approach: Functional characterization of these light-sensitive systems involves the use of biophysical, biochemical, genetic, microbiological and physiological approaches. Bacterial rhodopsin work requires measurement of light-driven ion translocation kinetics, stoichiometries, and quantum efficiencies in the intact cell and in cell membranes, or in purified pigments reconstituted into lipid vesicles. Cell swimming behavior and phototaxis are studied using computerized infrared video tracking techniques. Typically LOV domain proteins are cloned from specific organisms and expressed heterologously in E. coli and purified for biophysical, biochemical and photochemical characterization. In addition to conventional biochemical procedures for protein purification and sequencing, we use a variety of methods to assay light activation of enzymatic activities and molecular interactions, and DNA binding and/or gene expression regulation activities. When possible we establish phenotypes and study the regulation of biological activities in bacterial knockouts devoid of a particular LOV domain gene and in mutants complemented with native or specifically mutated genes. Testing of light regulation of virulence in pathogens is carried out in collaboration with groups that have the necessary bio-safety controls and facilities in the USA and abroad. Structural, spectroscopic and computational techniques used to characterize the chromoproteins, their chromophores, and their photochemical reactions include mass spectrometry, x-ray crystallography, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), absorption and fluorescence spectroscopy; laser flash photolysis in the ultraviolet, visible, and IR ranges; linear and circular dichroism; resonance Raman and Fourier transform infrared spectroscopy; and computational simulations of vibrational and electronic states and transitions. These studies are carried out in the native pigments and in chemically and genetically modified forms produced in the laboratory. |
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| (c) Roberto Bogomolni 2014 |