R. Scott McIsaac, Austin Rice, Claire Bedbrook, Lukas Herwig
Microbial opsin proteins, when expressed in neuronal cells and activated with light, turn ion flux on/off with millisecond speed. This allows us to control neuronal circuits with high spatial and temporal specificity, a capability that has revolutionized neuroscience. 'Optogenetic' research today depends upon a limited set of natural microbial opsins with broad activation spectra, limited ion selectivity and a narrow range of kinetics. The field needs a diverse population of engineered opsins, each with distinct properties for interrogating different neuronal pathways in different cell types. We are developing these new protein-based optogenetic tools.
Left: Opsins can be used to dynamically control neuronal activity in live animals, enabling researchers to assess the phenotypic consequences of stimulating/repressing particular neuronal circuits. Studying how groups of neurons work collectively will require opsins with shifted and narrowed activation spectra to enable multiplexed interrogation of these complex systems. Image source: http://www.stanford.edu/group/dlab/optogenetics/.
Engineering Actuators for Optogenetics
The Arnold lab is collaborating with the Gradinaru lab at Caltech to engineer microbial channelrhodopsins (ChRs), a family of retinylidene proteins that function as light-gated ion channels that induce neuronal depolarization when light-activated. Our goal is to engineer ChR proteins with novel cation specificity, narrow activation spectra, multicolor-light sensitivity and varied channel kinetics. These tools will facilitate optogenetic studies of healthy as well as impaired brain function, enabling targeted interrogation of the neural circuits that contribute to disorders such as Parkinson's disease, autism, schizophrenia, drug abuse and depression. In addition to many applications, our engineering efforts will improve our understanding of the sequence- and structure-function relationships for properties of interest.
Above: Contiguous SCHEMA recombination of channelrhodopsin. Different colors represent amino acid blocks that are swapped among functionally diverse opsins. The goal is to experimentally test the effects of these blocks on opsin functionality and use mathematical modeling to predict chimeras with desired properties.
Spectral Tuning of a Proton-Pumping Rhodopsin
Using directed evolution, we recently succeeded in tuning the absorption spectrum of a proton-pumping rhodopsin from Gloeobacter violaceus (called GR) by +/- 80 nm relative to the wild type protein. This study revealed that blue- and red-tuning mutations arise through different mechanisms: blue-tuning mutations change the polarity of the residues surrounding retinal, while red-tuning mutations disrupt the salt bridge between the protonated Schiff base and a nearby negatively charged residue (referred to as the negative counter-ion). What are the limits of spectral tuning? Can different non-retinal cofactors (i.e., unnatural analogs) be used to tune optical properties to generate useful proteins? Can we identify functional variants that are more shifted than variants found in nature? These are questions we are actively investigating.
Above: Spectral tuning of GR using directed evolution. Absorbances are color-coded according the colors of the resulting rhodopsin pigments.
Engineering Physiological Sensors
Opsins can also be used as fluorescent sensors that enable optical detection of changes in local electronic properties such as pH and voltage. Whereas opsins found in nature are very dim (quantum yields of ~10-4), we have found that directed evolution is an effective strategy for increasing fluorescence by nearly two orders of magnitude. For example, we increased the quantum yield of GR and Archaerhodopsin-3 (Arch) to >1%, meaning that >1% of all absorbed photons are emitted as fluorescence. These proteins emit fluorescence in the near-infrared, a desirable property for imaging deep into animal tissues. We have shown that a subset of our engineered Arch variants can be used as genetically encoded voltage indicators in C. elegans and cultured mammalian neurons. These tools should enable researchers to link changes in neuronal activity to behavioral phenotypes. Many questions remain. What are the limits of opsin fluorescence? Can we engineer variants that emit fluorescence at different wavelengths? What is the relationship between absorption, fluorescence, voltage sensitivity, and ion-pumping activity?
Above: Comparing fluorescence of wild-type Arch and an engineered variant, Arch(D95E/T99C).
Papers to Get Started
Directed Evolution of a Far-Red Fluorescent Rhodopsin. R. S. McIsaac, M. K. M. Engqvist, T. Wannier, A. Z. Rosenthal, L. Herwig, N. C. Flytzanis, E. S. Imasheva, J. K. Lanyi, S. P. Balashov, V. Gradinaru, F. H. Arnold. Proceedings of the National Academy of Sciences, early edition (2014). doi: 10.1073/pnas.1413987111
Directed Evolution of Gloebacter violaceus Rhodopsin Spectral Properties. M. K. M. Engqvist, R. S. McIsaac, P. Dollinger, N. C. Flytzanis, M. Abrams, S. Schor, F. H. Arnold. Journal of Molecular Biology in press, accepted article available online (2014). doi:10.1016/j.jmb.2014.06.015
Archaerhodopsin Variants with Enhanced Voltage Sensitive Fluorescence in Mammalian and C. elegans Neurons. N. C. Flytzanis, C. N. Bedbrook, H. Chiu, M. K. M. Engqvist, C. Xiao, K. Y. Chan, P. W. Sternberg, F. H. Arnold, V. Gradinaru. Nature Communications, in press.