Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization.

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments.

The use of amphipols for NMR structural characterization of 7-TM proteins.

While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.