[NMR paper] Optimizing the ?1B-adrenergic receptor for solution NMR studies.

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Optimizing the ?1B-adrenergic receptor for solution NMR studies.

Optimizing the ?1B-adrenergic receptor for solution NMR studies.

Biochim Biophys Acta Biomembr. 2020 May 12;:183354

Authors: Schuster M, Deluigi M, Panti? M, Vacca S, Baumann C, Scott DJ, Plückthun A, Zerbe O

Abstract
Sample preparation for NMR studies of G-protein coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes 13C, 15N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the ?1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini are removed and the intracellular loop 3 (IC3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1-2 mg/L of D2O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the ?1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [15N,1H]- as well as [13C,1H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [13C,1H]-HSQC spectra indicates that the so prepared receptor could be used for side-chain dynamics studies.


PMID: 32413443 [PubMed - as supplied by publisher]



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