Transferability of thermostabilizing mutations between β-adrenergic receptors

Abstract

In previous work we described six point mutations that thermostabilised the turkey β1-adrenergic receptor (tß1AR). The thermostable mutant, tß1AR-m23, had an apparent Tm 21°C higher than the native protein when solubilized in dodecylmaltoside (DDM) and, in addition, was significantly more stable in short chain detergents, which allowed its crystallization and structure determination. Identification of thermostabilizing mutations in tß1AR was performed by systematic mutagenesis followed by expressing and assaying each of the 318 mutants for their thermostability. This is time-consuming, so to facilitate studies on related receptors, we have studied the transferability of these mutations to the human adrenergic receptors, hß1AR and hß2AR, which have, respectively, 76% and 59% sequence identity to tß2AR, excluding the N- and C-termini. Thermostability assays revealed that hß1AR was much more unstable than tß2AR, whereas hß2AR was more stable than tß1 AR. Addition of the 6 thermostabilizing mutations in tß2AR-m23 into both hß2AR and hß2AR increased their apparent Tms by 17°C and 11°C, respectively. In addition, the mutations affected the global conformation of the human receptors so that they were predominantly in the antagonist bound form, as was originally observed for tß2AR- m23. Thus, once thermostabilizing mutations have been identified in one G protein-coupled receptor, stabilization of close members within the subfamily is rapidly obtainable. © 2009 Informa UK Ltd.