, Blue light sensor from algae, plants, bacteria and 633 funghi

, Bifunctional ligand containing a Ni 2+ -nitrilotriacetic 636 acid (Ni-NTA) moiety for selective coordination tethering to his-tagged receptors

. Maleimide, 1H-pyrrole-2,5-dione. Cysteine-reactive chemical group

, NM, 2-nitro-N-methylimidazolyl. Masking group selectively unmasked with NTR

, Nitroreductase from E. Coli used for selective reduction of NM groups

. Opto-xr, chimeric photocontrollable receptor engineered using opsins and the intracellular 645 loops or N-terminal tail of mammalian GPCRs

, Orthogonal, which does not interfere with native biological processes

. Peg, Flexible polymer that is highly water soluble

. Ple, Exogenous enzyme that efficiently and selectively hydrolyses CM 652 ester substrates

, Photoswitchable ligand 655 tethered to a protein or nanobody through SNAP-or CLIP-tag conjugation, PORTL, photoswitchable orthogonal remotely tethered ligand

. Psam/psem, Synthetic protein (pharmacologically selective actuator module, PSAM) that is 658 selectively activated by synthetic ligands (pharmacologically selective effector molecules, p.659

. Psems,

. Ptl, Thiol-reactive ligand incorporating a chemical 662 photoswitch, that photosensitizes cysteine-substituted receptors and ion channels

, Box 1: Bioconjugation technologies

, Bioconjugation reagents are used to link together a small chemical molecule

, It requires genetic modification of 753 the POI, in order to incorporate a reactive group that will serve as a biorthogonal handle for 754 conjugation. Multiple strategies exist [76]. The smallest and least disruptive genetic 755 modification is the incorporation of a cysteine amino acid on the protein surface through site-756 directed mutagenesis. Cysteines contain a thiol group that reacts efficiently, rapidly (minutes) 757 and with high selectivity with maleimide groups (Figure I) to form stable, p.758

, Cysteine has become the primary choice for site-specific modification of membrane 759 proteins because it is relatively low abundant, often engaged in disulfide bridges, and highly 760 nucleophilic at neural pH, vol.21

, cysteine-maleimide conjugation chemistry is restricted to extracellularly-762 accessible sites on membrane proteins [21]. In addition, because cysteines are naturally 763 present on many endogenous proteins, novel bioconjugation techniques that work inside cells 764 and that are fully bio-orthogonal have been developed. This includes the use of unnatural 765 amino acids (UAA) that contain a double (alkene) or triple bond (alkyne) for bioconjugation 766 with tetrazine-containing ligands through click chemistry [50] (Figure I). Click-chemistry is 767 extremely popular for protein bioconjugation because it relies on chemical groups that are 768 highly selective toward each other -yet remain inert otherwise-, exhibits fast reaction kinetics 769

. However, The other approaches for orthogonal labeling rely on larger 772 modifications of the POI, such as the incorporation of polypeptide tags. For instance, metal 773 chelation methods using poly-histidine tags (His-tag), which are classically used for protein 774 purification, have been used for non, vol.52

, conferring minimal disturbance to the protein, and label probes with high 776 efficiency and selectivity. Nevertheless, labeling is reversible and Ni is toxic to cells, hampering 777 in vivo use, His-tags are 775 small (4-9 residues), vol.76

, Finally, self-labeling domains such as SNAP-, CLIP-or HALO-tags use enzyme-catalyzed reactions for irreversible conjugation of ligands to POI in live cells. The reaction is 779 highly biorthogonal, rapid, irreversible and works intracellularly with low concentration of

, but requires fusion of the POI with a large protein domain 781 (>20 kDa) at the N-or C-terminus, which either is prohibited (as with nAChRs or GABA A Rs for 782 instance) or may affect POI function

, Box1 Figure I. Representative reactive groups for protein-ligand bioconjugation