Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location
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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 ~fourfold 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.
KeywordsMembrane proteins Sensitivity enhancement DNP Influenza M2 AMUPol Paramagnetic relaxation enhancement Solid-state NMR
The authors thank Prof. Robert Griffin, Eric Keeler and Vladmir Michaelis for stimulating discussions. This work is supported by National Institutes of Health Grants GM088204 and GM066976 to M.H.
- Cady SD, Wang J, Wu Y, DeGrado WF, Hong M (2011a) Specific binding of adamantane drugs and direction of their polar amines in the pore of the influenza M2 transmembrane domain in lipid bilayers and dodecylphosphocholine micelles determined by NMR spectroscopy. J Am Chem Soc 133:4274–4284CrossRefGoogle Scholar
- Franks WT, Zhou DH, Wylie BJ, Money BG, Graesser DT, Frericks HL, Sahota G, Rienstra CM (2005) Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis. J Am Chem Soc 127:12291–12305CrossRefGoogle Scholar
- Jacso T, Franks WT, Rose H, Fink U, Broecker J, Keller S, Oschkinat H, Reif B (2012) Characterization of membrane proteins in isolated native cellular membranes by dynamic nuclear polarization solid-state NMR spectroscopy without purification and reconstitution. Angew Chem Int Ed Engl 51:432–435CrossRefGoogle Scholar
- Rosay M, Tometich L, Pawsey S, Bader R, Schauwecker R, Blank M, Borchard PM, Cauffman SR, Felch KL, Weber RT, Temkin RJ, Griffin RG, Maas WE (2010) Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results. Phys Chem Chem Phys 12:5850–5860CrossRefGoogle Scholar