Orthogonal Optical Control of a G Protein-Coupled Receptor with a SNAP-Tethered Photochromic Ligand

Abstract

The covalent attachment of synthetic photoswitches is a general approach to impart light sensitivity onto native receptors. It mimics the logic of natural photoreceptors and significantly expands the reach of optogenetics. Here we describe a novel photoswitch design—the photoswitchable orthogonal remotely tethered ligand (PORTL)—that combines the genetically encoded SNAP-tag with photochromic ligands connected to a benzylguanine via a long flexible linker. We use the method to convert the G protein-coupled receptor mGluR2, a metabotropic glutamate receptor, into a photoreceptor (SNAG-mGluR2) that provides efficient optical control over the neuronal functions of mGluR2: presynaptic inhibition and control of excitability. The PORTL approach enables multiplexed optical control of different native receptors using distinct bioconjugation methods. It should be broadly applicable since SNAP-tags have proven to be reliable, many SNAP-tagged receptors are already available, and photochromic ligands on a long leash are readily designed and synthesized.

Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors

“…6F) (Broichhagen et al, 2015), similar to what is observed with MAG. We used a BGAG variant, BGAG 12,460 , which, following maximal labeling, acts as a full agonist when bound, but provides partial activation due to incomplete photoisomerization (Broichhagen et al, 2015) and, thus, should produce a population with a mix of receptors where 0, 1, or 2 subunits are liganded within a dimer. Thus, photoswitch efficiency relative to saturating glutamate should provide a measure of the relative activation efficiency of partially liganded dimers.…”

“…Because these mutants expressed poorly combined with the L300C substitution, we turned to an alternative approach for attachment of the photoswitchable glutamate ligand in which to an N-terminal SNAP domain is covalently labeled by benzylguanine-azobenzene-glutamate (“BGAG”) to enable photo-activation in cis (Fig. 6F) (Broichhagen et al, 2015), similar to what is observed with MAG. We used a BGAG variant, BGAG 12,460 , which, following maximal labeling, acts as a full agonist when bound, but provides partial activation due to incomplete photoisomerization (Broichhagen et al, 2015) and, thus, should produce a population with a mix of receptors where 0, 1, or 2 subunits are liganded within a dimer.…”

Precise modulation of neuronal activity with synthetic photoswitchable ligands

“…SNAP-tag) fused to a target receptor[52]. The complimentary switches, called PORTLs (photoswitchable orthogonal remotely tethered ligands), covalently bind to the self-labeling protein via selective chemistry (e.g.…”

“…a benzylguanine group in the case of SNAP). The technique was applied to a SNAP-mGluR2 fusion labeled with a glutamate-containing PORTL, making it possible to optically hyperpolarize and silence hippocampal neurons[52]. Perhaps the most exciting advantage with the addition of a new orthogonal chemical labeling strategy is the ability to individually express, label, and control more than one protein with light.…”

Molecular tools for acute spatiotemporal manipulation of signal transduction

“…In the photoswitchable tethered ligand (PTL) approach, these azobenzene-based photochromic ligands are attached to its receptor via maleimide conjugation to an engineered cysteine residue, resulting in a photoactivatable receptor. Broichhagen et al built upon this approach by fusing a GPCR with a SNAP tag coordinated with a photochromic ligand attached to a long flexible linker (Figure 2b) [35]. This method, called photoswitchable orthogonal remotely tethered ligand (PORTL), overcomes the limitations of maleimide-cysteine chemistry, such as nonspecific labeling as well as incompatibility with the intracellular environment.…”