Solid-State NMR Spectroscopy of Oriented Membrane Polypeptides at 100 K with Signal Enhancement by Dynamic Nuclear Polarization

Salnikov, Evgeniy S.; Rosay, Mélanie; Pawsey, Shane; Ouari, Olivier; Tordo, Paul; Bechinger, Burkhard

doi:10.1021/ja1007646

Cited by 76 publications

(63 citation statements)

References 27 publications

(63 reference statements)

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“…Thus, our preliminary DNP results at cryogenic temperature demonstrate that it is feasible to acquire reasonably well-resolved MAS spectra if the isotopically labeled sites in the embedded protein of interest are carefully chosen. Well-resolved MAS spectra along with determination of structural constraints have been demonstrated by the recent DNP solid-state NMR studies on samples including globular proteins [57,59,80], membrane proteins [15,48,49,51,53,55,58-62,82], cell extracts [27,77,83] and cellular samples [28,29]. Although the loss of spectral resolution limits the application of DNP MAS experiments at cryogenic temperatures, the sensitivity gain can be well utilized to measure high-resolution structural information if the system under study is selectively labeled with isotopes.…”

Section: Resultsmentioning

confidence: 99%

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Yamamoto

Caporini

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can be applied towards the determination of 3D structural information. However, there are numerous challenges that need to be overcome to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges with a specific emphasis on obtaining high-resolution structural insights into electron transfer biological processes mediated by membrane-bound proteins like mammalian cytochrome b5, cytochrome P450 and cytochrome P450 reductase. In this study, we demonstrate the feasibility of using the signal-enhancement rendered by dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement (ε). Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrates that it is highly possible to suppress the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution 3D structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.

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Section: Resultsmentioning

confidence: 99%

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Yamamoto

Caporini

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…bTbK (1) and bCTbK (4) are soluble in many organic solvents and they have been used successfully for DNP surface enhanced NMR spectroscopy (SENS) [14,23,24] and in preliminary solid-state NMR/DNP studies on membrane proteins [25]. TOTAPOL is soluble in glycerol/water (60/40) and is used to investigate biological systems [26][27][28], its efficiency is attributed to the existence of favored conformers exhibiting a somewhat serendipitous optimal orientation of nitroxide moieties for DNP.…”

Section: Compoundmentioning

confidence: 99%

Dinitroxides for Solid State Dynamic Nuclear Polarization

Ysacco¹,

Karoui²,

Casano³

et al. 2012

Appl Magn Reson

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International audienceWe have prepared a series of dinitroxides and we investigated theirproperties as polarizing agents for solid-state nuclear magnetic resonance/dynamicnuclear polarization applications at 100 K, 9.34 T (263 GHz electron paramagneticresonance and 400 MHz 1H nuclear magnetic resonance). Our results show that arigid structure with an orthogonal relative orientation of electron g tensors and theappropriate orientation of the two N O bonds are required to obtain maximumpolarization enhancements. In addition, with dinitroxides exhibiting a long T1e, thesaturation of the irradiated electron spin packet is favored leading to more efficientdynamic nuclear polarization

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“…Whole cells, cell envelopes, and native E. coli membranes enriched in specific membrane proteins showed enhancement factors of 20–30 (Jacso et al, 2012; Renault et al, 2012). 15 N NMR spectra of oriented membranes without cryoprotectant showed an enhancement factor of ~18 (Salnikov et al, 2010). …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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 ~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.

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Solid-State NMR Spectroscopy of Oriented Membrane Polypeptides at 100 K with Signal Enhancement by Dynamic Nuclear Polarization

Cited by 76 publications

References 27 publications

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Dinitroxides for Solid State Dynamic Nuclear Polarization

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

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