Abstract
The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a β turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the β turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.
Highlights
The defining feature of the ␣ subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys192 and Cys-193
Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a  turn to another backbone hydrogen bond, and across the subunit interface to the side chain of a functionally important Asp residue in the non-␣ subunit
We propose that the role of the vicinal disulfide is to distort the  turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp
Summary
Preparation of Cysteine Analogs—Cysteine analogs were prepared as 2-nitrobenzyl (NB) protected thiols. N-Me cysteine (370 mg, 2.7 mmol), prepared as described previously [27], was added to the vial, and the mixture was stirred for 10 min. The oil was purified by flash column chromatography on silica gel (50% EtOAC in hexanes) to afford N-Me, N-NVOC-Cys-(SNB)OCH2CN as a yellow oil in 62% yield (37 mg, 0.067 mmol). After cooling to room temperature, the mixture was concentrated in vacuo, and the resulting yellow oil was purified by flash column chromatography on silica gel (25% EtOAc in hexanes) to afford HO-Cys(SNB)-OMe in 35% yield (370 mg, 1.4 mmol). The pH of the aqueous layer was decreased to 1 via the addition of 6 M HCl and extracted with EtOAc. The new organic layer was washed with brine, dried over Na2SO4, and concentrated to yield HO-Cys(SNB)-OH as a yellow oil in 85% crude yield (350 mg, 1.4 mmol). Calculations on simple cyclic amides and related structures were performed with SPARTAN [37]
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