Fractional deuteration applied to biomolecular solid-state NMR spectroscopy

Nand, Deepak; Cukkemane, Abhishek; Becker, Stefan; Baldus, Marc

doi:10.1007/s10858-011-9585-2

Cited by 22 publications

(20 citation statements)

References 75 publications

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“…28 Note that all serine and threonine residues, which are the only amino acids that show intraresidual correlations in this region, have been previously assigned. 17,29 Since the asolectin lipids were not 13 C-labeled, the lipid cross-peaks are indicative for the presence of tightly bound lipids, copurified from the E. coli inner membrane.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Structural Determinants of Specific Lipid Binding to Potassium Channels

et al. 2013

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We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsA-Kv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while KcsA activity is critically modulated by the specific and cooperative binding of anionic nonannular lipids close to the channel's selectivity filter, the influence of nonannular lipid binding on KcsA-Kv1.3 is much reduced. The diminished impact of specific lipid binding on KcsA-Kv1.3 results from a point-mutation at the corresponding nonannular lipid binding site leading to a salt-bridge between adjacent KcsA-Kv1.3 subunits, which is conserved in many voltage-gated potassium channels and prevents strong nonannular lipid binding to the pore domain. Our findings elucidate how protein-lipid and protein-protein interactions modulate K(+) channel activity. The combination of MD, NMR, and functional studies as shown here may help to dissect the structural and dynamical processes that are critical for the functioning of larger membrane proteins, including Kv channels in a membrane setting.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Structural Determinants of Specific Lipid Binding to Potassium Channels

et al. 2013

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“…Experimental spectra were recorded on fully 13 C, 15 N-labeled ( Fig. S1) and on fractionally deuterated Chim (27). Resonance assignments were largely taken from published work (22).…”

Section: Resultsmentioning

confidence: 99%

Importance of lipid–pore loop interface for potassium channel structure and function

Cruijsen

Nand

Weingarth

et al. 2013

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

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Potassium (i.e., K + ) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K + channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K + channel inactivation and exhibits a remarkable structural plasticity that correlates to K + channel inactivation. The transmembrane helix 1 unwinds when the K + channel enters the inactivated state and rewinds during the transition to the closed state. In addition to wellcharacterized changes at the K + ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K + channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K + channel pore. membrane protein | ion channel | solid-state NMR spectroscopy P otassium (i.e., K + ) channels are embedded in the plasma membrane to control the selective passage of potassium ions across the lipid bilayer. The channels open and close their conduction pathway by sensing changes in physicochemical parameters such as pH, ligand concentration, and membrane voltage (1). Structure-function studies on voltage-gated K + (Kv) channels suggested that lipid molecules are an integral part of the voltage-sensing domains, which transfer during the gating process electrical charges across the cell membrane (2-4). In some of the available Kv channel crystal structures, lipid molecules appear most densely packed against the pore domain, presumably providing an appropriate environment for the stability and the operation of the gating machinery to open and close the conduction pathway. In general, the activity of Kv pore domains is thought to be determined by the activity of two gates in series, one for activation and one for inactivation. These gates jointly control the conduction of ions through the pore (5-11). The activation gate is located at the intracellular entrance of the pore and the inactivation gate is situated toward the extracellular entrance at the selectivity filter (i.e., C-type inactivation). In addition, some potassium channels possess close to the activation gate a receptor for an N-terminal inactivating domain (i.e., N-type inactivation).The K + channel pore domain is conserved across all K + channels. It comprises a tetrameric assembly of t...

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“…Furthermore, tailored labeling schemes can be used at the input stage 2 of FANDAS. So far, (1,3-13 C)-glycerol labeling and (2-13 C)-glycerol labeling (LeMaster and Kushlan 1996;Hong and Jakes 1999;Castellani et al 2002), fractional deuteration (Rosen et al 1996;Nand et al 2012), and 13 C, 15 N reverse labeling (Vuister et al 1994;Etzkorn et al 2007) have been implemented. Scrambling can be included as exemplified below.…”

Section: Fandas Modules and Program Usementioning

confidence: 99%

Rapid prediction of multi-dimensional NMR data sets

et al. 2012

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We present a computational environment for Fast Analysis of multidimensional NMR DAta Sets (FANDAS) that allows assembling multidimensional data sets from a variety of input parameters and facilitates comparing and modifying such "in silico" data sets during the various stages of the NMR data analysis. The input parameters can vary from (partial) NMR assignments directly obtained from experiments to values retrieved from in silico prediction programs. The resulting predicted data sets enable a rapid evaluation of sample labeling in light of spectral resolution and structural content, using standard NMR software such as Sparky. In addition, direct comparison to experimental data sets can be used to validate NMR assignments, distinguish different molecular components, refine structural models or other parameters derived from NMR data. The method is demonstrated in the context of solid-state NMR data obtained for the cyclic nucleotide binding domain of a bacterial cyclic nucleotide-gated channel and on membrane-embedded sensory rhodopsin II. FANDAS is freely available as web portal under WeNMR ( http://www.wenmr.eu/services/FANDAS ).

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Fractional deuteration applied to biomolecular solid-state NMR spectroscopy

Cited by 22 publications

References 75 publications

Structural Determinants of Specific Lipid Binding to Potassium Channels

Structural Determinants of Specific Lipid Binding to Potassium Channels

Importance of lipid–pore loop interface for potassium channel structure and function

Rapid prediction of multi-dimensional NMR data sets

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