Tetraphenylporphyrin derivative specifically blocks members of the voltage-gated potassium channel subfamily Kv1.

Tetraphenylporphyrin derivatives represent a promising class of high-affinity ligands for voltage-gated potassium (Kv) channels. Herein, we investigated the mode of Kv channel block of one tetraphenylporphyrin derivative, por3, using electrophysiological methods, structure-based mutagenesis, and solid-state NMR spectroscopy. The combined data showed that por3 specifically blocks Kv1.

Rapid prediction of multi-dimensional NMR data sets.

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.

Supramolecular structure of membrane-associated polypeptides by combining solid-state NMR and molecular dynamics simulations.

Elemental biological functions such as molecular signal transduction are determined by the dynamic interplay between polypeptides and the membrane environment. Determining such supramolecular arrangements poses a significant challenge for classical structural biology methods. We introduce an iterative approach that combines magic-angle spinning solid-state NMR spectroscopy and atomistic molecular dynamics simulations for the determination of the structure and topology of membrane-bound systems with a resolution and level of accuracy difficult to obtain by either method alone.

Protein refolding is required for assembly of the type three secretion needle.

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.

Amyloid-like interactions within nucleoporin FG hydrogels.

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 N-terminal part and passivates the FG hydrogel against nonselective interactions.

Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity.

Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting.

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

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.

Comparative analysis of NMR chemical shift predictions for proteins in the solid phase.

A comparative analysis of nuclear chemical shift predictions of proteins in the solid state by rapid algorithms trained on and verified with solution-state NMR assignments is presented. The precision of predictions by four dedicated computer programs (SHIFTS, PROSHIFTS, SHIFTX and SPARTA) was found to be close to values obtained for proteins in solution. Correlation coefficients depend on the NMR nucleus (N, C', C(alpha) and C(beta)) and on secondary structure (beta-strand, random coil and alpha-helix), but also on the molecular environment (membrane-integral or not).

Structural rearrangements of membrane proteins probed by water-edited solid-state NMR spectroscopy.

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.

A structural link between inactivation and block of a K+ channel.

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.

Solid-state NMR spectroscopy applied to a chimeric potassium channel in lipid bilayers.

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.

Solid-state 31P NMR spectroscopy of precipitated guanine nucleotide-binding protein Ras in complexes with its effector molecules Raf kinase and RalGDS.

Liquid-state 31P NMR spectroscopy is a well-established method for the study of guanine nucleotide-binding proteins (GNB proteins) such as the proto-oncogene Ras. Solid-state 31P NMR spectroscopy could meanwhile also be used to study microcrystalline samples of Ras as well as its partial loss-of-function mutants Ras(T35S) and Ras(T35A). However, solid-state NMR studies of the latter mutants in complex with effector molecules such as RalGDS or Raf kinase were so far prevented, since it has been impossible to crystallize these complexes yet.

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy.

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein.