Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry

Abstract

Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP efficiency. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T 1 relaxation, microwave on versus off signal intensities and DNP enhancement. The data are suggestive that for membranes the speed of cooling has a pronounced effect on membrane phase transitions and concomitantly the distribution of biradicals within the sample.

Metal‐Chelated Polymer Nanodiscs for NMR Studies

“…This system coupled with recent developments in our lab to measure RDCs will yield an useful tool for fast NMR acquisition in the study of biomolecules as well as the already known application in MP research. This study also creates potential avenues to use the paramagnetic nature of the chelated polymer nanodiscs for dynamic nuclear polarization (DNP) solid‐state NMR experiments to overcome the sensitivity issues in studying membrane proteins. We expect that the reported novel polymer design would enable multi‐labeling to utilize the benefits of PRE, 19 F and DNP approaches for distance measurements on membrane proteins by solid‐state NMR spectroscopy.…”

Metal‐Chelated Polymer Nanodiscs for NMR Studies

“…Secondly, the facile polymer preparation allows for exploitation of the paramagnetic effects of metal ions complexed by nanodiscs without the need for costly metal complexed lipids. This strategy would also enable the use of PRE in the structural studies of membrane proteins …”

“…Secondly,t he facile polymer preparation allows for exploitation of the paramagnetic effects of metal ions complexed by nanodiscs without the need for costly metal complexed lipids.T his strategy would also enable the use of PRE in the structural studies of membrane proteins. [28,31,32] Here we report the synthesis of am odified poly(styreneco-maleic acid) (SMA) derivative called SMA-EA-DOTA which is engineered with am etal chelator and forms lipidnanodiscs.S MA-EA-DOTA polymer was synthesized similarly to SMA-EA polymer,w hich has been previously reported to form stable nanodiscs and is used as acomparative system in this study.T osynthesize the polymer,weused alow molecular weight (M n % 1600 gmol À1 )poly(styrene-co-maleic anhydride) (SMAnh) as the starting material. [19] Thechemical modification of the SMAnh includes on average one DOTA chelator molecule per polymer chain modification ( Figure 1a; see methods section in the Supporting Information).…”

“…This system coupled with recent developments in our lab to measure RDCs [23] will yield an useful tool for fast NMR acquisition in the study of biomolecules as well as the already known application in MP research. This study also creates potential avenues to use the paramagnetic nature of the chelated polymer nanodiscs for dynamic nuclear polarization (DNP) solid-state NMR experiments [31,32,[42][43][44][45] to overcome the sensitivity issues in studying membrane proteins.W e expect that the reported novel polymer design would enable multi-labeling to utilize the benefits of PRE, 19 Fa nd DNP approaches for distance measurements on membrane proteins by solid-state NMR spectroscopy. Figure 5.…”

PHRONESIS: A One‐Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid‐State NMR Spectroscopy

“…[1][2][3][4][5][6] The past few years have witnessed a significant effort to speed up the ssNMR experiments using ultrafast magic angle spinning (MAS), dynamic nuclear polarization (DNP), paramagnetic relaxation enhancement (PRE), ultra-high-field magnets, and 1 H detected experiments. [7][8][9][10][11][12][13][14][15][16][17][18] Additionally, the introduction of multi-acquisition polarization optimized experiments (POE) has further boosted data acquisition [19][20][21][22][23] for both solution-and ssNMR spectroscopy. [19][20][21][22][23][24][25][26][27][28][29][30][31][32] Indeed, 1 H detection has significantly improved the sensitivity of fast and ultrafast MAS experiments for fully protonated protein samples; however, the broad line widths of the 1 H resonances reduce the spectral resolution.…”