Mapping disulfide connectivity using backbone ester hydrolysis.
The site-specific incorporation of alpha-hydroxy acids into proteins using nonsense suppression can provide a powerful probe of protein structure and function. The resulting backbone ester may be selectively hydrolyzed in the presence of the peptide backbone, providing an "orthogonal" chemistry that can be useful both as an analytical tool and as a structural probe. Here we describe in detail a substantial substituent effect on this hydrolysis reaction. Consistent with mechanistic expectations, the steric bulk of the amino acid immediately N-terminal of the hydroxy acid has a large effect on the hydrolysis rate. On the basis of these results, we also describe a simple protocol for identifying disulfide loops in soluble and membrane proteins, exemplified by the alpha subunit of the muscle nicotinic acetylcholine receptor (nAChR). If a backbone ester is incorporated outside a disulfide loop, hydrolysis alone gives two fragments, but if the ester is incorporated within a disulfide loop, both hydrolysis and reduction are required for cleavage. This test could be useful in characterizing the disulfide topology of complex, membrane proteins.
England
P M
PM
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Lester
H A
HA
Dougherty
D A
DA
D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004952:Esters; D005260:Female; D006868:Hydrolysis; D006880:Hydroxy Acids / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D017354:Point Mutation; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D004220:Disulfides; D004952:Esters; D006880:Hydroxy Acids; D010446:Peptide Fragments; D011978:Receptors, Nicotinic