Site-specific, photochemical proteolysis applied to ion channels in vivo

Abstract

A method for site-specific, nitrobenzylinduced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K ؉ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the ''signature'' Cys128-Cys142 disulfide loop of the nAChR ␣ subunit.A powerful tool for probing protein structure and function is the incorporation of unnatural amino acids by the method of nonsense suppression, a technique developed for in vitro translation systems by Schultz and coworkers (1-3). Recent work has expanded the scope of this methodology by establishing procedures for unnatural amino acid incorporation into proteins expressed in living cells (4-7). This allows the evaluation of neuroreceptors, ion channels, and related proteins that are studied most effectively in vivo using heterologous expression systems such as the Xenopus oocyte. The suppression methodology is especially promising for such integral membrane proteins, which are not yet generally amenable to the methods of high resolution structure determination (e.g., NMR, x-ray crystallography).In this paper we (i) describe the unnatural amino acid 2-(nitrophenyl)glycine (Npg) and show that it can be efficiently incorporated into proteins expressed in vivo; (ii) establish that in vivo irradiation of an Npg-containing protein leads to site-specific, nitrobenzyl-induced photochemical proteolysis (SNIPP) (Fig. 1); and (iii) illustrate the usefulness of this residue by cleavage of the Cys128-Cys142 ''signature'' disulfide loop of the nicotinic acetylcholine receptor, establishing a crucial functional role for this structural feature.The design of the unnatural amino acid Npg was based on the photochemistry of 2-nitrobenzyl derivatives. Compounds of this type, including Npg itself, have been used as protecting groups in organic synthesis and to produce ''caged'' neurotransmitters, ions, and second messengers that can then be liberated photochemically, but, to our knowledge, Npg has not been incorporated into peptides or proteins (8-17). As shown in Fig. 1, incorporation of Npg into a protein, followed by irradiation of the protein, was anticipated to produce peptide backbone cleavage (18) dures (19-20).-(2-nitrophenyl)glycine was protected as the N-pent-4-enoyl (4PO) derivative using standard conditions (21, 22). Na 2 CO 3 (111 mg, 1.05 mmol) was added to a room temperature solution of (2-nitrophenyl)glycine hydrochloride (82 mg, 0.35 mmol) in H 2 O:dioxane (0.75 ml:0.5 ml), followed by a solution of pent-4-enoic anhydride (70.8 mg, ...

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

“…The "all-genetic" strategy, which uses engineered orthogonal aaRS/suppressor tRNA pairs, has significantly enhanced biological and technical convenience (8,28). We envision further development in the genetic encoding of various photoreactive UAAs into LGICs, such as photo-caged amino acids (26,(37)(38)(39), or UAAs containing azo-benzene moieties (7) that adopt two conformations with different wavelengths. Incorporation of the latter would provide the possibility of generating bidirectionally controlled LGICs, which could be switched between on and off states in response to light, as afforded by photoswitchable ligands (1,2).…”

Photochemical Proteolysis of an Unstructured Linker of the GABAAR Extracellular Domain Prevents GABA but Not Pentobarbital Activation

“…Npg-tRNA was prepared as described previously (England et al, 1997). Nitrophenylglycine was prepared according to published procedures (Davis et al, 1973;Muralidharan and Nerbonne, 1995;England et al, 1997).…”

“…In the present work, we disrupt this linker region and evaluate the impact on agonist-mediated receptor function in the ␣ 1 ␤ 2 GABA A R. In particular, we use nonsense suppression (Nowak et al, 1995(Nowak et al, , 1998 to site-specifically incorporate the photoresponsive unnatural amino acid nitrophenylglycine (Npg) into this linker (England et al, 1997). Exposure to UV light then initiates photochemical cleavage of the GABA A R backbone via a well precedented mechanism (Scheme 1).…”

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

“…Similarly, specific cleavage at aminooxyacetic acid can be achieved with zinc and acetic acid, and cleavage of the protein backbone at allylglycine is possible upon treatment with iodine [204,205]. Photochemical proteolysis occurs upon irradiation at 2-nitrophenylglycines [206]. Photocaged groups, such as nitrobenzyl ethers and esters, shield reactive moieties until irradiative deprotection, and can be used to control enzymatic activity or protein-protein interaction in a time-resolved manner [207][208][209].…”