Optimizing the α1B-adrenergic receptor for solution NMR studies

Matthias Schuster, Mattia Deluigi, Milica Pantić, Santiago Vacca, Christian Baumann, Daniel J Scott, Andreas Plückthun, Oliver Zerbe
2020
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
more » ... 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 were removed and the intracellular loop 3 (ICL3) 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 studying side-chain dynamics. 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 13 C, 15 N, 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 α 1B -adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1-2 mg/L of D 2 O 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 α 1B -adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [ 15 N, 1 H]-as well as [ 13 C, 1 H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [ 13 C, 1 H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.
doi:10.5167/uzh-187976 fatcat:bz72wptqljcchnezxji6hwhaua