Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 413, Issue 3, Pages 628-638Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2011.08.057
Keywords
G-protein-coupled receptor; membrane protein; thermostabilisation
Categories
Funding
- Commonwealth Scholarship
- Medical Research Council [MC_U105197215] Funding Source: researchfish
- MRC [MC_U105197215] Funding Source: UKRI
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Conformational thermostabilisation of G-protein-coupled receptors is a successful strategy for their structure determination. The thermostable mutants tolerate short-chain detergents, such as octylglucoside and nonylglucoside, which are ideal for crystallography, and in addition, the receptors are preferentially in a single conformational state. The first thermostabilised receptor to have its structure determined was the beta(1)-adrenoceptor mutant beta(1)AR-m23 bound to the antagonist cyanopindolol, and recently, additional structures have been determined with agonist bound. Here, we describe further stabilisation of beta(1)AR-m23 by the addition of three thermostabilising mutations (I129V, D322K, and Y343L) to make a mutant receptor that is 31 degrees C more thermostable than the wild-type receptor in dodecylmaltoside and is 13 degrees C more thermostable than beta(1)AR-m23 in nonylglucoside. Although a number of thermostabilisation methods were tried, including rational design of disulfide bonds and engineered zinc bridges, the two most successful strategies to improve the thermostability of beta(1)AR-m23 were an engineered salt bridge and leucine scanning mutagenesis. The three additional thermostabilising mutations did not significantly affect the pharmacological properties of beta(1)AR-m23, but the new mutant receptor was significantly more stable in short-chain detergents such as heptylthioglucoside and denaturing detergents such as SDS. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.
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