A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

Abstract

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4 . Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.

Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes

“…Herein we describe a molecular rationale for a sample preparation strategy utilizing nitroxide radical labeled lipids (spin labeled lipids, SL-lipids, Figure 1) that we recently demonstrated gave large dynamic nuclear polarization (DNP) NMR signal enhancements for membrane embedded peptides. 3 …”

“…17 For heterogeneous biological macromolecular ssNMR samples such as membrane proteins, fibrils, and macromolecular assemblies, DNP enhancement gradients have been observed, with largest enhancement values observed for nuclei that are located at the interface between the aqueous phase and the molecule of interest with diminishing values obtained for nuclei located within the sample interior. 3, 23, 24 For example, with the use of TOTAPOL or AMUPol in membrane protein DNP MAS ssNMR studies, the largest enhancement values are observed at the hydrophilic periphery of the membrane and the least enhancement is observed at the hydrophobic interior of the membrane, where the bulk of the protein is embedded thus demonstrating a distance dependence on polarization transfer efficiency. 3 This observed enhancement gradient is attributed to the mechanism of polarization transfer in the DNP experiment.…”

“…3, 23, 24 For example, with the use of TOTAPOL or AMUPol in membrane protein DNP MAS ssNMR studies, the largest enhancement values are observed at the hydrophilic periphery of the membrane and the least enhancement is observed at the hydrophobic interior of the membrane, where the bulk of the protein is embedded thus demonstrating a distance dependence on polarization transfer efficiency. 3 This observed enhancement gradient is attributed to the mechanism of polarization transfer in the DNP experiment. Specifically, polarization is first induced by the paramagnetic agent in local nuclei, which are NMR silent due to relaxation effects that originate from their close proximity to the paramagnetic species.…”

“…3 Several noteworthy advantages of the SL-lipid polarizing agents were demonstrated. (1) By locating the nitroxide within the lipid milieu, a more homogeneous distribution of DNP enhancements was observed throughout the various carbon nuclei of the phospholipids.…”

Solid-state NMR: An emerging technique in structural biology of self-assemblies

“…A major issue in MAS ssNMR is obtaining high quality spectra (good signal-to-noise ratio, high spectral resolution). The growing repertoire of polarization transfer schemes (reviewed in [68][69][70][71]), access to high magnetic fields (up to 1 GHz), the development of fast to ultra-fast MAS probes [72][73][74] and associated methods [75,76], the development of efficient rotor filling procedures [77,78], the use of specific labeling schemes [79][80][81][82][83], the development of proton detected experiments [74,[84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101], and biomolecular dynamic nuclear polarization [60,[102][103][104][105][106][107][108][109][110][111][112]<...>…”