Engineering an ultra-thermostable β(1)-adrenoceptor.

Journal of molecular biology (2011-09-13)
Jennifer L Miller, Christopher G Tate

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 β(1)-adrenoceptor mutant β(1)AR-m23 bound to the antagonist cyanopindolol, and recently, additional structures have been determined with agonist bound. Here, we describe further stabilisation of β(1)AR-m23 by the addition of three thermostabilising mutations (I129V, D322K, and Y343L) to make a mutant receptor that is 31 °C more thermostable than the wild-type receptor in dodecylmaltoside and is 13 °C more thermostable than β(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 β(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 β(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.

Product Number
Product Description

Pindolol, ≥98% (TLC), powder