Nanosculpting reversed wavelength sensitivity into a photoswitchable iGluR

Abstract

Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: (i) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and (ii) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparation, whereas a second wavelength has the opposite effect. The ability to generate opposite responses with a single PTL and 2 versions of a target channel, which can be expressed in different cell types, paves the way for engineering opponency in neurons that mediate opposing functions.glutamate receptor ͉ ion channel ͉ optics ͉ photoswitch

Optochemical Genetics

“…[69] In another case of "sign inversion," we found that the point of attachment can determine whether a PTL activates the channel in the cis or in the trans form of the photoswitch (Figure 13). [69] MAG-0 bound to L439C, our original attachment site, acts as a cis agonist but becomes a trans agonist when attached to L486C. This is somewhat surprising given that these two sites are only a few ngstroms apart in the Xray structures of GluK2.…”

“…[68,69] LiGluR shows some fundamental functional differences compared with SPARK. Both irradiation and depolarization are needed to open the SPARK channel (corresponding to a logic AND gate).…”

Structural Optimization of Photoswitch Ligands for Surface Attachment of α‐Chymotrypsin and Regulation of Its Surface Binding

“…The dialkyne 23 [27] was treated with TMSCl to give the monoprotected alkyne 24 in 26 % yield. Treatment of 24 with the bromoazobenzene 16 in the presence of [PdA C H T U N G T R E N N U N G (PPh 3 ) 2 Cl 2 ] and CuI was attempted, but the desired product 26 was not obtained, possibly due to homocoupling of the alkyne 24.…”

“…The trifluoromethyl ketone 3 and the a-keto ester 7 were each prepared by means of a Sonogashira coupling reaction as the key step (Scheme 2). The bromoazobenzene 16 [23] was coupled to 2-methylbut-3-yn-2-ol in the presence of [Pd-A C H T U N G T R E N N U N G (PPh 3 ) 2 Cl 2 ] to give the alkyne 17 in 93 % yield. Both protecting groups were removed on hydrolysis with aqueous NaOH to give 18, which was separately treated with 10 and with the a-hydroxy ester 20 [24] in the presence of EDCI as in similar syntheses [25] to give the amides 19 and 21, respectively.…”

“…[1,2] The incorporation of this functionality into a (photo)switchable surface, for example, shows much promise for the development of "intelligent" nanoscale biosystems. [3] Photoresponsive surfaces that are able to modulate the binding and release of molecules (an enzyme, for example) in reversible fashion offer potential for the development of new biosensors, advanced medical implants, biomolecular computers, and advanced protein purification procedures based on photoaffinity chromatography.…”