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Long-Range Coupling in an Allosteric Receptor Revealed by Mutant Cycle Analysis

Gleitsman, Kristin R. and Shanata, Jai A. P. and Frazier, Shawnalea J. and Lester, Henry A. and Dougherty, Dennis A. (2009) Long-Range Coupling in an Allosteric Receptor Revealed by Mutant Cycle Analysis. Biophysical Journal, 96 (8). pp. 3168-3178. ISSN 0006-3495. PMCID PMC2718292. doi:10.1016/j.bpj.2008.12.3949. https://resolver.caltech.edu/CaltechAUTHORS:20090827-132541833

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Abstract

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50–60 Å from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (βL9′S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC50 measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bpj.2008.12.3949DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718292/PubMed CentralArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2009 by the Biophysical Society. Received 18 September 2008; accepted 15 December 2008. We thank B. N. Cohen for advice on single-channel recording and analysis. This work was supported by the National Institutes of Health (NS 34407; NS 11756). J.A.P.S. was partially supported by a National Research Service Award training grant. K.R.G. was partially supported by a National Science Foundation Graduate Research Fellowship.
Funders:
Funding AgencyGrant Number
NIHNS 34407
NIHNS11756
NSFUNSPECIFIED
Issue or Number:8
PubMed Central ID:PMC2718292
DOI:10.1016/j.bpj.2008.12.3949
Record Number:CaltechAUTHORS:20090827-132541833
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20090827-132541833
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15360
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:10 Sep 2009 22:54
Last Modified:08 Nov 2021 23:18

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