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Probing Hydronium Ion Histidine NH Exchange Rate Constants in the M2 Channel via Indirect Observation of Dipolar-Dephased 15N Signals in Magic-Angle-Spinning NMR

Fu, Riqiang, Miao, Yimin, Qin, Huajun, Cross, Timothy A.
Journal of the American Chemical Society 2016 v.138 no.49 pp. 15801-15804
histidine, ions, nitrogen, nuclear magnetic resonance spectroscopy, pH, proteins, protons, stable isotopes
Water–protein chemical exchange in membrane-bound proteins is an important parameter for understanding how proteins interact with their aqueous environment, but has been difficult to observe in membrane-bound biological systems. Here, we demonstrate the feasibility of probing specific water–protein chemical exchange in membrane-bound proteins in solid-state MAS NMR. By spin-locking the ¹H magnetization along the magic angle, the ¹H spin diffusion is suppressed such that a water–protein chemical exchange process can be monitored indirectly by dipolar-dephased ¹⁵N signals through polarization transfer from ¹H. In the example of the Influenza A full length M2 protein, the buildup of dipolar-dephased ¹⁵N signals from the tetrad of His37 side chains have been observed as a function of spin-lock time. This confirms that hydronium ions are in exchange with protons in the His37 NH bonds at the heart of the M2 proton conduction mechanism, with an exchange rate constant of ∼1750 s–¹ for pH 6.2 at −10 °C.