Transferability of thermostabilizing mutations between beta-adrenergic receptors

Abstract

In previous work we described six point mutations that thermostabilised the turkey beta(1)-adrenergic receptor (tbeta(1)AR). The thermostable mutant, tbeta(1)AR-m23, had an apparent T(m) 21 degrees 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 tbeta(1)AR 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, hbeta(1)AR and hbeta(2)AR, which have, respectively, 76% and 59% sequence identity to tbeta(2)AR, excluding the N- and C-termini. Thermostability assays revealed that hbeta(1)AR was much more unstable than tbeta(2)AR, whereas hbeta(2)AR was more stable than tbeta(1)AR. Addition of the 6 thermostabilizing mutations in tbeta(2)AR-m23 into both hbeta(2)AR and hbeta(2)AR increased their apparent T(m)s by 17 degrees C and 11 degrees 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 tbeta(2)AR-m23. Thus, once thermostabilizing mutations have been identified in one G protein-coupled receptor, stabilization of close members within the subfamily is rapidly obtainable.