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 ~110Â*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 105â??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 ~10Â*Ã? 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.
[NMR paper] Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins.
Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins.
Related Articles Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins.
J Magn Reson. 2013 Nov 28;239C:9-15
Authors: Tesch DM, Nevzorov AA
Abstract
Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample...
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12-21-2013 03:15 PM
[NMR paper] Sensitivity Enhancement and Contrasting Information Provided by Free Radicals in Oriented-Sample NMR of Bicelle-Reconstituted Membrane Proteins
Sensitivity Enhancement and Contrasting Information Provided by Free Radicals in Oriented-Sample NMR of Bicelle-Reconstituted Membrane Proteins
Publication date: Available online 28 November 2013
Source:Journal of Magnetic Resonance</br>
Author(s): Deanna M. Tesch , Alexander A. Nevzorov</br>
Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents.Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological...
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11-28-2013 05:18 PM
NMR Sample Preparation from NESG Wiki
NMR Sample Preparation from NMR2.0 Wiki
http://www.nmr2.buffalo.edu/nesg.wiki/NMR_Sample_Preparation
originally posted by Main.Gaohua.Liu (11 Feb 2007)
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08-16-2010 04:04 AM
Sample preparation
Sample preparation Samples
The Samples program is designed to collect and store some essential information pertaining to the samples used to record the spectra.
Residual Dipolar Couplings
Bicelle preparation notes from Avanti
Instructions for bicelle preparation from Avanti