Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy

Ader, Christian; Pongs, Olaf; Becker, Stefan; Baldus, Marc

doi:10.1016/j.bbamem.2009.06.023

Cited by 38 publications

(30 citation statements)

References 67 publications

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“…2 D, right), possibly due to local motion. Previously, we detected such increased mobility of selectivity filter residues, in particular for V76, in ssNMR studies using synthetic (45) as well as native bacterial membranes (46,47), in line with results of previous molecular-dynamics studies (48).…”

Section: Resultssupporting

confidence: 90%

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Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Cruijsen

Prokofyev

Pongs

et al. 2017

Biophysical Journal

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Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K þ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K þ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.

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Section: Resultssupporting

confidence: 90%

“…(22)) for E71 corroborated our previous analysis of the chimeric KcsA-Kv1.3 channel, which differs from the WT KcsA in 11 amino acids in the turret region (45). In both cases, the collapsed state of the selectivity filter (SF) involves protonation of E71 (Fig.…”

Section: Resultssupporting

confidence: 87%

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Cruijsen

Prokofyev

Pongs

et al. 2017

Biophysical Journal

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“…Since typical longitudinal relaxation-rate constants of 15 N and 13 C are in the order of seconds, high-power proton irradiation of several seconds would be required in order to saturate the protons in solids. This is experimentally impossible with current probe designs and to our knowledge all measurements of longitudinal relaxation-rate constants in solid proteins have been done without proton saturation during the relaxation delay [96,98,99,[104][105][106]. This is justified by the action of proton spin diffusion in solids that leads to an effect that is very similar to proton saturation.…”

Section: Longitudinal Relaxation Parameters In Solid-state Nmrmentioning

confidence: 99%

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

Schanda

Ernst

2016

Progress in Nuclear Magnetic Resonance Spectroscopy

182

292

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Magic-angle spinning solid-state NMR spectroscopy is an important technique to study molecular structure, dynamics and interactions, and is rapidly gaining importance in biomolecular sciences. Here we provide an overview of experimental approaches to study molecular dynamics by MAS solid-state NMR, with an emphasis on the underlying theoretical concepts and differences of MAS solid-state NMR compared to solution-state NMR. The theoretical foundations of nuclear spin relaxation are revisited, focusing on the particularities of spin relaxation in solid samples under magic-angle spinning. We discuss the range of validity of Redfield theory, as well as the inherent multi-exponential behavior of relaxation in solids. Experimental challenges for measuring relaxation parameters in MAS solid-state NMR and a few recently proposed relaxation approaches are discussed, which provide information about time scales and amplitudes of motions ranging from picoseconds to milliseconds. We also discuss the theoretical basis and experimental measurements of anisotropic interactions (chemical-shift anisotropies, dipolar and quadrupolar couplings), which give direct information about the amplitude of motions. The potential of combining relaxation data with such measurements of dynamically-averaged anisotropic interactions is discussed. Although the focus of this review is on the theoretical foundations of dynamics studies rather than their application, we close by discussing a small number of recent dynamics studies, where the dynamic properties of proteins in crystals are compared to those in solution.

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“…These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1)(2)(3)(4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

mentioning

confidence: 99%

“…A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, flux-dependent inactivation occurs in Na + and Ca 2+ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7,(13)(14)(15)(16)(17)(18).…”

mentioning

confidence: 99%

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Brettmann

Urusova

Tonelli

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassiumselective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.solution NMR | ion channels | membrane protein | protein dynamics

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Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy

Cited by 38 publications

References 67 publications

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

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