Expression and biophysical analysis of two double‐transmembrane domain‐containing fragments from a yeast G protein‐coupled receptor

Abstract

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on inclusion bodies. Nonlabeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation ([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis and the efficient CNBr processing and purification yields allowed the initiation of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was monomeric in this micellar environment, whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], but had a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-CT40 exhibited more than 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these large domains of a GPCR in micelles using high-resolution NMR.