期刊
MOLECULAR MEMBRANE BIOLOGY
卷 26, 期 8, 页码 385-396出版社
TAYLOR & FRANCIS LTD
DOI: 10.3109/09687680903208239
关键词
Membrane protein; G protein-coupled receptor; conformational thermostabilization
资金
- Pfizer Global Research and Development
- MRCT Development Gap Fund [A853-0031]
- MRC
In previous work we described six point mutations that thermostabilised the turkey beta(1)-adrenergic receptor (t beta(1)AR). The thermostable mutant, t beta(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 us crystallization and structure determination Identification of thermostabilizing mutations in t beta(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, h beta(1)AR and h beta(2)AR, which have, respectively, 76% and 59% sequence identity to t beta(2)AR, excluding the N- and C-termini. Thermostability, assays revealed that h beta(1)AR was much more unstable than t beta(2)AR, whereas h beta(2)AR was more stable than t beta(1)AR Addition of the 6 thermostabilizing mutations in t beta(2)AR-m23 into both h beta(2)AR and h beta(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 t beta(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.
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