Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

Abstract

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel

“…2c–e). A sensitivity enhancement factor of 21–27 was achieved using a recently optimized membrane preparation protocol for DNP NMR 41 . With the removal of the natural abundance 13 C signals of lipids and cryoprotectants, we resolved two His19 Cβ signals at 29 and 27 ppm and the S12 Cα and Cβ signals at ~60 ppm (Fig.…”

“…BM2(1–33) was reconstituted into a deuterated lipid mixture denoted d 31 -VM+’, which contains d 31 -POPC, d 31 -POPE and cholesterol at a molar ratio of 40% : 40% : 20% 41 . The partially deuterated phospholipids were used to increase the efficiency of polarization transfer from the radical to the peptide.…”

“…The peptide-lipid mixture was suspended in a 10 mM pH 5.5 citrate buffer and centrifuged at 40,000 rpm at 4°C overnight to obtain a homogeneous pellet, which was equilibrated to a hydration level of ~40 wt%. A small amount of a stock DNP solution, d 8 -glycerol : D 2 O : H 2 O (60 : 30 : 10 by volume) containing 10 mM of the biradical AMUPol was titrated into the membrane pellet 41 . The pellet was vortexed to distribute the radical uniformly, and D 2 O was added to reach a D 2 O : H 2 O volume ratio of 3 : 1.…”

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

“…This can be achieved by searching for different sample preparation protocols rather than using the established glycerol matrix (Liao et al 2016;Takahashi et al 2013) and by searching for biradicals that allow reasonable enhancement factors at higher temperatures (Lelli et al 2015;Perras et al 2016).…”

Multidimensional solid-state NMR spectroscopy of plant cell walls

“…In the new single-network model, cellulose has limited interactions with hemicellulose but extensive contacts with pectins. To understand how this single polysaccharide network is loosened during plant cell growth, we employed 2D correlation experiments under dynamic nuclear polarization (DNP) [40–42] to determine the binding target of expansin [32, 43] in Arabidopsis cell walls. A bacterial analog of the plant expansin [44] was expressed in Escherichia coli and enriched with 13 C and 15 N, while the cell wall is enriched only in 13 C. Using a 13 C- 15 N dipolar filter, we selected the protein 13 C polarization, then allowed it to transfer to the adjacent polysaccharides.…”