Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

NMR studies of a channel protein without membranes: structure and dynamics of water-solubilized KcsA.

ABSTRACT: Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics.

SUBMITTER: Ma D

PROVIDER: S-EPMC2572164 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

NMR studies of lipid regulation of the K<sup>+</sup> channel KcsA.

Project description:The membrane environment, including specific lipid characteristics, plays important roles in the folding, stability, and gating of the prokaryotic potassium channel KcsA. Here we study the effect of membrane composition on the population of various functional states of KcsA. The spectra provide support for the previous observation of copurifying phospholipids with phosphoglycerol headgroups. Additional, exogenously added anionic lipids do not appear to be required to stabilize the open conductive conformation of KcsA, which was previously thought to be the case. On the contrary, NMR-based binding studies indicate that including anionic lipids in proteoliposomes at acidic pH leads to a weaker potassium ion affinity at the selectivity filter. Since K+ ion loss leads to channel inactivation, these results suggest that anionic lipids promote channel inactivation.

| S-EPMC9189731 | biostudies-literature

Potassium channel, ions, and water: simulation studies based on the high resolution X-ray structure of KcsA.

Project description:Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.

| S-EPMC1303560 | biostudies-literature

Resurrecting abandoned proteins with pure water: CD and NMR studies of protein fragments solubilized in salt-free water.

Project description:Many proteins expressed in Escherichia coli cells form inclusion bodies that are neither refoldable nor soluble in buffers. Very surprisingly, we recently discovered that all 11 buffer-insoluble protein fragments/domains we have, with a great diversity of cellular function, location, and molecular size, could be easily solubilized in salt-free water. The circular dichroism (CD) and NMR characterization led to classification of these proteins into three groups: group 1, with no secondary structure by CD and with narrowly-dispersed but sharp (1)H-(15)N heteronuclear single quantum correlation (HSQC) peaks; group 2, with secondary structure by CD but with HSQC peaks broadened and, consequently, only a small set of peaks detectable; and group 3, with secondary structure by CD and also well-separated HSQC peaks. Intriguingly, we failed to find any protein with a tight tertiary packing. Therefore, we propose that buffer-insoluble proteins may lack intrinsic ability to reach or/and to maintain a well-packed conformation, and thus are trapped in partially-folded states with many hydrophobic side chains exposed to the bulk solvent. As such, a very low ionic strength is sufficient to screen out intrinsic repulsive interactions and, consequently, allow the hydrophobic clustering/aggregation to occur. Marvelously enough, it appears that in pure water, proteins have the potential to manifest their full spectrum of structural states by utilizing intrinsic repulsive interactions to suppress the attractive hydrophobic clustering. Our discovery not only gives a novel insight into the properties of insoluble proteins, but also sheds the first light that we know of on previously unknown regimes associated with proteins.

| S-EPMC1635667 | biostudies-literature

Local and global structure of the monomeric subunit of the potassium channel KcsA probed by NMR.

Project description:KcsA is a homotetrameric 68-kDa membrane-associated potassium channel which selectively gates the flux of potassium ions across the membrane. The channel is known to undergo a pH-dependent open-to-closed transition. Here we describe an NMR study of the monomeric subunit of the channel (KcsAM), solubilized in SDS micelles. Chemical shift, solvent exchange, backbone 15N relaxation and residual dipolar coupling (RDC) data show the TM1 helix to remain intact, but the TM2 helix contains a distinct kink, which is subject to concentration-independent but pH-dependent conformational exchange on a microsecond time scale. The kink region, centered at G99, was previously implicated in the gating of the tetrameric KcsA channel. An RDC-based model of KcsAM at acidic pH orients TM1 and the two helical segments of the kinked TM2 in a configuration reminiscent of the open conformation of the channel. Thus, the transition between states appears to be an inherent capability of the monomer, with the tetrameric assembly exerting a modulatory effect upon the transition which gives the channel its physiological gating profile.

| S-EPMC2174260 | biostudies-literature

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.

Project description:As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

| S-EPMC9250420 | biostudies-literature

Functional equilibrium of the KcsA structure revealed by NMR.

Project description:KcsA is a tetrameric K(+) channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p(perm)) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1-3% 2,2,2-trifluoroethanol increases p(perm) and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.

| S-EPMC3501015 | biostudies-literature

(17)O NMR Investigation of Water Structure and Dynamics.

Project description:The structure and dynamics of the bound water in barium chlorate monohydrate were studied with (17)O nuclear magnetic resonance (NMR) spectroscopy in samples that are stationary and spinning at the magic-angle in magnetic fields ranging from 14.1 to 21.1 T. (17)O NMR parameters of the water were determined, and the effects of torsional oscillations of the water molecule on the (17)O quadrupolar coupling constant (CQ) were delineated with variable temperature MAS NMR. With decreasing temperature and reduction of the librational motion, we observe an increase in the experimentally measured CQ explaining the discrepancy between experiments and predictions from density functional theory. In addition, at low temperatures and in the absence of (1)H decoupling, we observe a well-resolved (1)H-(17)O dipole splitting in the spectra, which provides information on the structure of the H2O molecule. The splitting arises because of the homogeneous nature of the coupling between the two (1)H-(17)O dipoles and the (1)H-(1)H dipole.

| S-EPMC5418864 | biostudies-literature

Anionic phospholipid interactions with the potassium channel KcsA: simulation studies.

Project description:Molecular dynamics (MD) simulations have been used to unmask details of specific interactions of anionic phospholipids with intersubunit binding sites on the surface of the bacterial potassium channel KcsA. Crystallographic data on a diacyl glycerol fragment at this site were used to model phosphatidylethanolamine (PE), or phosphatidylglycerol (PG), or phosphatidic acid (PA) at the intersubunit binding sites. Each of these models of a KcsA-lipid complex was embedded in phosphatidyl choline bilayer and explored in a 20 ns MD simulation. H-bond analysis revealed that in terms of lipid-protein interactions PA > PG >> PE and revealed how anionic lipids (PG and PA) bind to a site provided by two key arginine residues (R(64) and R(89)) at the interface between adjacent subunits. A 27 ns simulation was performed in which KcsA (without any lipids initially modeled at the R(64)/R(89) sites) was embedded in a PE/PG bilayer. There was a progressive specific increase over the course of the simulation in the number of H-bonds of PG with KcsA. Furthermore, two specific PG binding events at R(64)/R(89) sites were observed. The phosphate oxygen atoms of bound PG formed H-bonds to the guanidinium group of R(89), whereas the terminal glycerol H-bonded to R(64). Overall, this study suggests that simulations can help identify and characterize sites for specific lipid interactions on a membrane protein surface.

| S-EPMC1367107 | biostudies-literature

Conformational dynamics of the KcsA potassium channel governs gating properties.

Project description:K+ channels conduct and regulate K+ flux across the cell membrane. Several crystal structures and biophysical studies of tetrameric ion channels have revealed many of the structural details of ion selectivity and gating. A narrow pore lined with four arrays of carbonyl groups is responsible for ion selectivity, whereas a conformational change of the four inner transmembrane helices (TM2) is involved in gating. We used NMR to examine full-length KcsA, a prototypical K+ channel, in its open, closed and intermediate states. These studies reveal that at least two conformational states exist both in the selectivity filter and near the C-terminal ends of the TM2 helices. In the ion-conducting open state, we observed rapid structural exchange between two conformations of the filter, presumably of low and high K+ affinity, respectively. Such measurements of millisecond-timescale dynamics reveal the basis for simultaneous ion selection and gating.

| S-EPMC3525321 | biostudies-literature

Computational design of water-soluble analogues of the potassium channel KcsA.

Project description:Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield in Escherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.

| S-EPMC357012 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data