High-frequency dynamic nuclear polarization in MAS spectra of membrane and soluble proteins.
One of the principal promises of solid-state NMR (SSNMR) magic angle spinning (MAS) experiments has been the possibility of determining the structures of molecules in states that are not accessible via X-ray or solution NMR experiments-e.g., membrane or amyloid proteins. However, the low sensitivity of SSNMR often restricts structural studies to small-model compounds and precludes many higher-dimensional solid-state MAS experiments on such systems. To address the sensitivity problem, we have developed experiments that utilize dynamic nuclear polarization (DNP) to enhance sensitivity. In this communication, we report the successful application of MAS DNP to samples of cryoprotected soluble and membrane proteins. In particular, we have observed DNP signal enhancements of up to 50 in 15N MAS spectra of bacteriorhodopsin (bR) and alpha-lytic protease (alpha-LP). The spectra were recorded at approximately 90 K where MAS is experimentally straightforward, and the results suggest that the described protocol will be widely applicable.
Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Melanie
M
Rosay
Jonathan C
JC
Lansing
Kristin C
KC
Haddad
William W
WW
Bachovchin
Judith
J
Herzfeld
Richard J
RJ
Temkin
Robert G
RG
Griffin
D001436:Bacteriorhodopsins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012697:Serine Endopeptidases / Q000737:chemistry*; D012995:Solubility
D001436:Bacteriorhodopsins; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase