The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based, sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain, which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the Nterminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular β-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain β-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage.Beta-sheet | nuclear pore complex | Nup | Prion | solid-state NMR N uclear pore complexes (NPCs) control all nucleocytoplasmic exchange (1-4). Their permeability barrier allows free passage of small molecules but suppresses the flux of macromolecules larger than 30 kDa and thereby prevents an uncontrolled intermixing of nuclear and cytoplasmic contents. However, the permeability barrier also permits a rapid passage of even large cargoes, provided these are bound to appropriate nuclear transport receptors (NTRs) (2-5). NTRs thereby supply nuclei with proteins and the cytoplasm with ribosomes and other nuclear products.FG repeat domains are essential building blocks of NPCs (6). They are considered to be natively unfolded and contain up to 50 repeat units, in which a characteristic hydrophobic patch, typically with the sequence FG, FxFG, or GLFG, is surrounded by more hydrophilic spacer sequences (7-9). These hydrophobic patches transiently bind NTRs during facilitated NPC passage (10-15).Recent evidence suggests that the permeability barrier is a hydrogel derived from FG repeat domains (FG hydrogel) (13,(15)(16)(17)(18). FG hydrogels have been predicted by the selective phase model (15,16,19) and indeed have been reconstituted from the FG/FxFG domain of the yeast nucleoporin Nsp1p (13,16) or the GLFG domains from Nup49p and Nup57p (17). All these gels showed permeability properties very similar to those of NPCs themselves: they allowed an up to 20,000...

We show that water-edited solid-state NMR spectroscopy allows for probing global protein conformation and residue-specific solvent accessibility in a lipid bilayer environment. The transfer dynamics can be well described by a general time constant, irrespective of protein topology and lipid environment. This approach was used to follow structural changes in response to protein function in the chimeric potassium channel KcsA-Kv1.3. Data obtained as a function of pH link earlier biochemical data to changes in protein structure in a functional bilayer setting.

Gating the ion-permeation pathway in K(+) channels requires conformational changes in activation and inactivation gates. Here we have investigated the structural alterations associated with pH-dependent inactivation gating of the KcsA-Kv1.3 K(+) channel using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. Transition of the KcsA-Kv1.3 K(+) channel from a closed state at pH 7.5 to an inactivated state at pH 4.0 revealed distinct structural changes within the pore, correlated with activation-gate opening and inactivation-gate closing. In the inactivated K(+) channel, the selectivity filter adopts a nonconductive structure that was also induced by binding of a pore-blocking tetraphenylporphyrin derivative. The results establish a structural link between inactivation and block of a K(+) channel in a membrane setting.

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from -helix into -strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

Potassium (K þ )-channel gating is choreographed by a complex interplay between external stimuli, K þ concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K þ , pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K þ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K þ ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K þ -channel pore.

We show that solid-state NMR can be used to investigate the structure and dynamics of a chimeric potassium channel, KcsA-Kv1.3, in lipid bilayers. Sequential resonance assignments were obtained using a combination of (15)N- (13)C and (13)C- (13)C correlation experiments conducted on fully labeled and reverse-labeled as well as C-terminally truncated samples. Comparison of our results with those from X-ray crystallography and solution-state NMR in micelles on the closely related KcsA K (+) channel provides insight into the mechanism of ion channel selectivity and underlines the important role of the lipid environment for membrane protein structure and function.

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 ).

We report longitudinal (15)N relaxation rates derived from two-dimensional ((15)N, (13)C) chemical shift correlation experiments obtained under magic angle spinning for the potassium channel KcsA-Kv1.3 reconstituted in multilamellar vesicles. Thus, we demonstrate that solid-state NMR can be used to probe residue-specific backbone dynamics in a membrane-embedded protein. Enhanced backbone mobility was detected for two glycine residues within the selectivity filter that are highly conserved in potassium channels and that are of core relevance to the filter structure and ion selectivity.