EAPSI: Mechanistic Understanding of Light Sensitive Proteins for Biomedical Applications

Tachibana Sean R, Corvallis OR

Investigators

Abstract

Optogenetics is a biological method that genetically modifies living cells in order to use light to control molecular processes. Current biomedical and life sciences have benefited from the applications of optogenetic tools such as sensing analytes in the body (in vivo) like calcium, controlling ion channels for communication, and genome editing in hopes of curing genetic diseases. Optogenetic applications are currently limited by the specificity or accuracy in binding targeted molecules and the rate it takes to turn the tool on and off. Dr. Moritoshi Sato is one of the leading professors working on optogenetics at the University of Tokyo. Under the guidance of Dr. Sato and his team, the Fellow will receive hands-on experience in developing new photoswitches for optogenetic control along with making published photoswitches. Photoswitching proteins, naturally found in plants, fungi, and bacteria, have allowed protein engineers to take advantage of the optogenetic methods. Natural photoswitches are known to homodimerize and exhibit slow switch-off kinetics which decrease the efficiency of optical control and limit their applications in optogenetics. Dr. Moritoshi Sato at the University of Tokyo has created novel photoswitches named Magnets that were derived from a photoreceptor named Vivid. By engineering the cofactor-binding domain, the Sato lab was able to selectively induce fast heterodimerization and tune the switch-off kinetics from hours to seconds. These Magnets were implemented into a genome editing protein named CRISPR-Cas9 which was originally developed to be controlled chemically. The incorporation of Magnets led to a photoactivatable Cas9 (paCas9) which enables optogenetic control. The overarching goal of this project is to synthesize photoswitches (Vivid, Magnet variants, and paCas9) using Dr. Sato's protein engineering facility so the samples can be later analyzed using the ultrafast laser spectroscopy facility at Oregon State University. We will exploit the wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) technique to track the photocatalytic reaction pathways around the flavin adenine dinucleotide (FAD) chromophore in different Vivid mutants including the slow and fast Magnets. This work will provide a fundamental understanding of how the current mutations within the Vivid cofactor-binding domain give rise to specific changes in the protein function, aided by the sulfur-containing amino acid residues in the vicinity of FAD. As a result, structural dynamics basis for the Magnet light sensing efficiency, dimerization, and switch-on and off kinetics can be elucidated. The optimization of optogenetic tools will allow for advanced applications in neuron modulation, drug delivery, bioimaging, and biosensors. The integration of bioengineering and biophysics enables a bottom-up approach to fundamentally understand photochemistry at the chemical bond level for photoswitchable proteins, and to rationally design and effectively achieve the desired protein functionality. This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the Japan Society for the Promotion of Science.