Abstract
1H-detected solid-state NMR in combination with 1H/2D exchange steps allows for the direct identification of very strong hydrogen bonds in membrane proteins. On the example of the membrane-embedded potassium channel KcsA, we quantify the longevity of such very strong hydrogen bonds by combining time-resolved 1H-detected solid-state NMR experiments and molecular dynamics simulations. In particular, we show that the carboxyl-side chain of the highly conserved residue Glu51 is involved in ultra-strong hydrogen bonds, which are fully-water-exposed and yet stable for weeks. The astonishing stability of these hydrogen bonds is important for the structural integrity of potassium channels, which we further corroborate by computational studies.
Highlights
The measurement of hydrogen-deuterium (1H/2D) exchange is a widespread method in solution NMR to probe protein structure [1], folding [2], and protein-ligand interactions [3]
While the indirect 15N-detection of 1H/2D exchange could already be used for elegant solid-state NMR (ssNMR) studies on the topology of 7TM membrane proteins [5], we are likely to see many more applications of the simple 1H/2D exchange tool with the advent of high-resolution 1H-detected ssNMR [6], driven by fast magic angle spinning (MAS)
Note that the water-exposed cytoplasmic domain (CPD) is not visible in our dipolar-based spectra due to increased mobility, as we demonstrated previously [7b]
Summary
The measurement of hydrogen-deuterium (1H/2D) exchange is a widespread method in solution NMR to probe protein structure [1], folding [2], and protein-ligand interactions [3]. Its principle is straightforward and based on the signal attenuation of exchangeable protons such as amino or hydroxyl protons upon incubation with deuterated solvents. How fast these protons exchange is usually determined by their participation in hydrogen bonds and by their surface exposure. The latter aspect renders 1H/2D exchange naturally a very attractive method to study membrane protein topology, which has been exploited in a number of solution NMR studies in micelles or detergents [4]. In solid-state NMR (ssNMR), 1H/2D exchange could not be directly exploited due a lack of 1H-resolution. 1H-detected 1H/2D exchange ssNMR studies were conducted on the membrane protein proteorhodopsin [8] and on the SH3 domain [9]
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