Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Atomistic Understanding and Design of Microbial Opsin-based Optogenetics Tools

Photo-sensitive ion transports of microbial opsins with a bound chromophore have been utilized in optogenetics for optical control of neuronal activity. Microbial opsins are characterized by seven-transmembrane helices that bind a chromophore, retinal, to a lysine residue of the protein through a protonated Schiff base linkage. Rhodopsins, the opsin-retinal complexes, exhibit photo-absorption in the visible wavelength region which triggers the functional activities of ion transport across cell membrane. In optogenetics, microbial opsins that function as light-sensitive ion transporters are heterologously expressed in neurons to excite and silence them by illumination with light. The functionalities of microbial opsins in optogenetics have been diversified through genomic searches for analogous light-sensitive ion transporters and molecular engineering. The availability of their X-ray crystallographic structures also allows one to elucidate physico-chemical mechanisms of their functionalities and to design in atomistic details engineered variants for the use in optogenetics.

In this study, we performed theoretical analyses of functional properties of microbial rhodopsins employed in optogenetics as well as computational design of their mutational variants with new functionality. Various computational approaches including quantum chemistry, molecular dynamics simulations, and statistical integral equation theory are utilized in combinatorial ways to examine molecular mechanisms underlying functional properties such as color tuning, light-gating/switching, and ion transport, and to model mutational variants based on the mechanisms elucidated.

First, we attempted to create color variants of microbial rhodopsins which permit one to perform dual optical control of neuronal activity and simultaneous optical control/observation. Color of the bound retinal chromophore is very sensitive to electrostatic field of the protein surroundings and to its conformational distortion sterically imposed in the binding cavity. Understanding and designing color variants therefore require accurate structural modeling of engineered proteins which possibly undergo extensive conformational changes upon replacement of critical residues. Through molecular modeling with a recently developed hybrid molecular simulation technique called QM/MM RWFE-SCF (Kosugi & Hayashi, 2012) which allows one to take into account extensive conformational changes of proteins upon mutations yet with high accuracy of quantum chemistry description of the chromophore's structure, we succeeded in theoretical design of color variants of microbial rhodopsins which were experimentally confirmed to undergo large spectral shifts yet with keeping photo-sensitive ion transport activities.

We also carried out theoretical analysis of photo-sensitive ion conduction. Photo-activation of the protein was modelled by molecular dynamics simulations, and then distributions of water molecules and ions in the ion channel were examined by statistical integral equation theory. The analysis identifies the ion conduction pathway as well as the gate that controls the photo-sensitivity of the ion conduction.

    Kosugi, T. & Hayashi S. (2012) QM/MM reweighting free energy SCF for geometry optimization on extensive free energy surface of enzymatic reaction. J. Chem. Theory and Comput., 8, 322-334.

    Kosugi, T. & Hayashi S. (2012) Crucial role of protein flexibility in formation of a stable reaction transition state in an α-amylase catalysis. J. Am. Chem. Soc., 134, 7045-7055.

Shigehiko Hayashi

Department of Chemistry
Graduate School of Science
Kyoto University
Kyoto 606-8502, Japan

Ph: +81-75-753-4006
Fx: +81-75-753-4000