1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ |
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| Authors: | João Medeiros‐Silva Deni Mance Mark Daniëls Shehrazade Jekhmane Dr. Klaartje Houben Prof. Marc Baldus Dr. Markus Weingarth |
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| Affiliation: | NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Utrecht, The Netherlands |
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| Abstract: | 1H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1H 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‐resolution 1H‐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. |
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| Keywords: | in-cell NMR spectroscopy membrane proteins protein dynamics proton detection solid-state NMR spectroscopy |
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