Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization

Abstract

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.

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

“…Using DNP tagging strategies, one can envision tagging proteins in complex environments to achieve selective NMR enhancement by differential DNP enhancement of the target protein over background proteins [149,150]. As mentioned above, this selective detection is crucial for NMR studies in the cellular context, to prevent signals of other proteins in the environment from obscuring those of the protein of interest.…”

“…As mentioned above, this selective detection is crucial for NMR studies in the cellular context, to prevent signals of other proteins in the environment from obscuring those of the protein of interest. This was demonstrated in cell lysate for the protein Bcl-X L using a TOTAPOL derivatized peptide [150]. By subtracting the background signal (from a sample without the selective Bak peptide), researchers were able to collect DNP enhanced 2D spectra of Bcl-X L in cellular extracts prepared from cells grown in U ( 13 C, 15 N) media.…”

“…In this vein, Etzkorn and coworkers modified the Bak peptide with TOTAPOL [150] which binds with a K D of 340 nm to the protein Bcl-X L . They showed an enhancement of 18 for the bound protein.…”

“…In these studies the polarization buildup times suggested that the experiments achieved selective sensitization of the bound protein. In the studies involving Mn(II), transfer occurs directly to 13 C, and selective enhancements were achieved without deuteration of the matrix [149,170]. In E , conditions for selective enhancement for the tagged biradical TMP-T are illustrated, namely a well-deuterated matrix; when 20% protons are added to the solvent, the glycerol signals are enhanced greater than the background.…”

Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags

“…Recent attempts to control radical solubility and location include solubilization in surfactants, 27,28 caging in cyclodextrin, 29 labeling peptides with biradicals, 30 radical tagged lipids, 31,32 “gluing” with trehalose, 33 use of endogenous paramagnetic cofactors, 34,35 labeling with paramagnetic metal chelators, 36 and the use of a biradical-tagged peptide that binds the protein of interest. 37 Covalent attachment of the biradicals TOTAPOL and AMUPOL via cysteine-specific methanthiosulfonate chemistry has also been reported as a method for matrix-free membrane protein enhancement. 22,38 …”