Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes

Abstract

Dynamic nuclear polarization (DNP) enhanced solid-state NMR can provide orders of magnitude in signal enhancement. One of the most important aspects of obtaining efficient DNP enhancements is the optimization of the paramagnetic polarization agents used. To date, the most utilized polarization agents are nitroxide biradicals. However, the efficiency of these polarization agents is diminished when used with samples other than small molecule model compounds. We recently demonstrated the effectiveness of nitroxide labeled lipids as polarization agents for lipids and a membrane embedded peptide. Here, we systematically characterize, via electron paramagnetic (EPR), the dynamics of and the dipolar couplings between nitroxide labeled lipids under conditions relevant to DNP applications. Complemented by DNP enhanced solid-state NMR measurements at 600 MHz/395 GHz, a molecular rationale for the efficiency of nitroxide labeled lipids as DNP polarization agents is developed. Specifically, optimal DNP enhancements are obtained when the nitroxide moiety is attached to the lipid choline head group and local nitroxide concentrations yield an average e−-e− dipolar coupling of 47 MHz. Based on these measurements, we propose a framework for development of DNP polarization agents optimal for membrane protein structure determination.

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

“…Notably, the enhancement was higher than that seen for soluble TOTAPOL, and there was no need for exogenous glassing agents. In follow-up work [161], Smith et al showed that for lipid resonances, flexible monoradicals tagged to the membrane surface provided the best enhancements. In all of these examples, monoradicals are used to provide a source of polarization for cross effect DNP due to intermolecular radical interactions.…”

A quasi-optical and corrugated waveguide microwave transmission system for simultaneous dynamic nuclear polarization NMR on two separate 14.1 T spectrometers

“…Germane to this work is the development of two 14.1 T DNP systems: one supporting MAS-DNP experiments with a commercially available probe [24,25], and the other aimed at providing DNP enhancement of solution state samples via the Overhauser effect. Commercially available gyrotrons operating at 395 GHz, of the kind developed by Bruker Biospin in collaboration with Communications & Power Industries (CPI), generate microwave beams that are powerful enough to support DNP-enhanced NMR experiments on two 14.1 T NMR systems simultaneously.…”

Dynamic Nuclear Polarization/Solid‐State NMR Spectroscopy of Membrane Polypeptides: Free‐Radical Optimization for Matrix‐Free Lipid Bilayer Samples

“…Assuming an extended conformation of the fatty acyl chain within the hydrophobic region of the bilayers, the distance between the two unpaired electrons was controlled by the positioning of the doxyl nitroxide at different positions of the fatty acyl chains. The design aimed at an inter‐radical distance of roughly 10–12 Å, which corresponds to magnetic dipolar interactions of around 30–50 MHz by using the point‐dipole approximation …”

“…Using liposomal membrane models, the groups of De Paëpe and Long recently reported polarizing agents specifically designed for solid‐state NMR/DNP experiments, namely, palmitoyl–TOTAPOL and pairs of mononitroxide‐labeled lipids, respectively ,. Both studies provided useful and unique information and highlighted the challenge of getting DNP enhancements larger than 10 in phosphatidylcholine (PC) and 1,2‐dipalmitoyl‐ sn ‐glycero‐3‐phosphocholine/1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐ phosphocholine (DPPC/POPC) membrane systems.…”