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Mapping Disulfide Connectivity Using Backbone Ester Hydrolysis

England, Pamela M. and Lester, Henry A. and Dougherty, Dennis A. (1999) Mapping Disulfide Connectivity Using Backbone Ester Hydrolysis. Biochemistry, 38 (43). pp. 14409-14415. ISSN 0006-2960. doi:10.1021/bi991424c.

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The site-specific incorporation of α-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 α 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.

Item Type:Article
Related URLs:
URLURL TypeDescription
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 1999 American Chemical Society. Received June 22, 1999; Revised Manuscript Received August 20, 1999; Publication Date (Web): October 5, 1999. This work was supported by grants from the National Institutes of Health (NS 34407 and NS 11756).
Funding AgencyGrant Number
NIHNS 34407
NIHNS 11756
Issue or Number:43
Record Number:CaltechAUTHORS:20180313-080855191
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Official Citation:Mapping Disulfide Connectivity Using Backbone Ester Hydrolysis. Pamela M. England, Henry A. Lester, and Dennis A. Dougherty. Biochemistry 1999 38 (43), 14409-14415. DOI: 10.1021/bi991424c
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:85264
Deposited By: Tony Diaz
Deposited On:13 Mar 2018 17:40
Last Modified:15 Nov 2021 20:27

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