1H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In Vitro and In Situ.
1H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for1H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution1H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
João
J
Medeiros-Silva
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
Deni
D
Mance
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
Mark
M
Daniëls
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
Shehrazade
S
Jekhmane
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
Klaartje
K
Houben
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
Marc
M
Baldus
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands. M.H.Weingarth@uu.nl.
Markus
M
Weingarth
D001426:Bacterial Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry*; D066244:Proton Magnetic Resonance Spectroscopy; D014867:Water / Q000737:chemistry*
D001426:Bacterial Proteins; D008081:Liposomes; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D014867:Water
in-cell NMR spectroscopy; membrane proteins; protein dynamics; proton detection; solid-state NMR spectroscopy
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