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I design and study proteins with light controlled activity.

Protein engineering, optogenetics, coffee, analog photography


Optogenetics is the use of naturally occuring light sensing proteins to help study biology.


Too fast to follow. Many processes in biology occur so quickly or in such complex environments that our traditional methods of studying them are not sufficient. Imagine you’d like to investigate a process occurring in a mouse’s brain. If you administer a drug, it may be slow to start acting, and may not specifically target the region of interest. What if you could specifically control one region of the brain, even one cell, simply by shining a light on it?

From microbes to the brain. Specialized proteins called microbial opsins transport positive or negative ions across cell membranes in response to light. This action closely resembles the electrical signaling in the nervous system. When these proteins are delivered to neurons in the brain, they can be used activate or silence this signaling. This allows scientists to determine the function of specific neurons in the brain.

Stuck in the membrane? Light driven ion transport is a powerful technique, but is there a way to study processes away from the cell membrane, inside of the cell? As it turns out, there are many other light sensing proteins found in nature. For example, light sensing proteins influence growth towards sunlight in plants. By modifying these proteins, we can achieve light switchable control of other biological processes. This can be done by producing a library of many protein sequences and searching for the desired light-switchable activity, or by rationally engineering the new function by using the protein’s three dimensional structure as a guide.


Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration.

Abstract: Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein-coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions.

Pub.: 13 Jun '14, Pinned: 15 Apr '17

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.

Abstract: The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

Pub.: 24 Dec '14, Pinned: 15 Apr '17