Topical Developments in High‐Field Dynamic Nuclear Polarization

Michaelis, Vladimir K.; Ong, Ta‐Chung; Kiesewetter, Matthew K.; Frantz, Derik K.; Walish, Joseph J.; Ravera, Enrico; Luchinat, Claudio; Swager, Timothy M.; Griffin, Robert G.

doi:10.1002/ijch.201300126

Cited by 41 publications

(48 citation statements)

References 122 publications

(169 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We conducted these experiments at higher magnetic fields (700MHz rather than 211 MHz) to achieve significant improvements in spectral resolution (Barnes et al, 2012; Michaelis et al, 2014). We collected a one-bond 13 C- 13 C dipolar-assisted rotational resonance (DARR)(Takegoshi et al, 2001) correlation spectrum on 1 mg of cryoprotected, purified NM fibrils in six hours.…”

Section: Resultsmentioning

confidence: 99%

Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieus

Frederick

Michaelis

Corzilius

et al. 2015

Cell

Self Cite

134

157

View full text Add to dashboard Cite

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of NMR spectroscopy and enable structural studies in biologically complex environments. Here we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an instrinsically disordered region, the yeast prion protein Sup35. We added an exogenously-prepared isotopically-labeled protein to deuterated lysates, rendering the biological environment “invisible” and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible and such contexts can influence protein structure.

show abstract

Section: Resultsmentioning

confidence: 99%

Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieus

Frederick

Michaelis

Corzilius

et al. 2015

Cell

Self Cite

134

157

View full text Add to dashboard Cite

show abstract

“…The two-orders-of-magnitude enhancements are achieved in practice using a number of crucial elements: a paramagnetic polarizing agent in the form of a stable radical, a high-power and high-frequency microwave source (Bajaj et al, 2007; Barnes et al, 2008; Becerra et al, 1993; Gerfen et al, 1995; Rosay et al, 2010), and low temperature to slow down electron and nuclear spin relaxation. A wide variety of mono- and bi-radicals have been designed and synthesized (Kubicki et al, 2016; Michaelis et al, 2014), with the two most commonly used ones being TOTAPOL and AMUPol, which contain two nitroxide radicals separated by ~13 Å via intervening functional groups with varying lengths, rigidity and polarity (Hu et al, 2008; Hu et al, 2004; Sauvee et al, 2013; Song et al, 2006). At low temperatures of 90–120 K commonly used for DNP SSNMR experiments, a cryoprotecting solution is often used to distribute the exogenous radical uniformly in the sample and to prevent ice formation at low temperature in hydrated biological samples.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

“…As already mentioned, DNP has proven beneficial both in the case of proteic systems as in case of materials [154][155][156]. It has to be noted that DNP is usually performed at cryogenic temperatures (<100 K) in order to lengthen the electron relaxation time, thus relaxing the need for high-power microwave irradiation, as well as to mitigate the heating effects of microwave irradiation itself [157].…”

Section: Dnp Studies Of Biosilicamentioning

confidence: 99%