Mapping disulfide connectivity using backbone ester hydrolysis

The site-specific incorporation of a-hydroxy acids into proteins using nons ense suppression can provide a powerful probe of protein structure and func tion. The resulting backbone ester may be selectively hydrolyzed in the pre sence of the peptide backbone, providing an "orthogonal" chemistry that can be useful both as an analytical tool and as a structural probe. Here we de scribe in detail a substantial substituent effect on this hydrolysis reacti on. Consistent with mechanistic expectations, the steric bulk of the amino acid immediately N-terminal of the hydroxy acid has a large effect on the h ydrolysis rate. On the basis of these results, we also describe a simple pr otocol for identifying disulfide loops in soluble and membrane proteins, ex emplified by the alpha subunit of the muscle nicotinic acetylcholine recept or (nAChR). If a backbone ester is incorporated outside a disulfide loop, h ydrolysis alone gives two fragments, but if the ester is incorporated withi n a disulfide loop, both hydrolysis and reduction are required for cleavage . This test could be useful in characterizing the disulfide topology of com plex, membrane proteins.