Markus Weingarth

Efficient heteronuclear decoupling by quenching rotary resonance in solid-state NMR

0009-2614/

Solid-state NMR-based approaches for supramolecular structure elucidation.

see front matter 2008 Elsevier B.V. A doi:10.1016/j.cplett.2008.10.041 * Corresponding author. Fax: +33 144 323 344. E-mail address: Piotr.Tekely@ens.fr (P. Tekely). We propose a new scheme for heteronuclear decoupling designed for fast magic-angle spinning (MAS), dubbed phase-inverted supercycled sequence for attenuation of rotary resonance (PISSARRO). Its efficiency compares favourably with CW, TPPM, SPINAL and XiX decoupling methods at medium and high RF amplitudes, particularly under conditions where the efficiency of decoupling is affected by undesired rotary resonance effects. 2008 Elsevier B.V. All rights reserved.

Low-power decoupling at high spinning frequencies in high static fields.

Supramolecular chemistry provides structural and conformational information about complexes formed from multiple molecules. While the molecule is held together by strong intramolecular contacts like covalent bonds, supramolecular structures can be further stabilized by weaker or transient intermolecular interactions. These interactions can confer a great diversity and sensitivity to exogenous factors like temperature, pressure, or ionic strength to multimolecular arrangements. Solid-state nuclear magnetic resonance (ssNMR) can provide atomic-scale structural and dynamical information in highly disordered or heterogeneous biological systems, even in complex environments such as cellular membranes or whole cells. In these systems, the molecule of interest no longer exists as a separate unit, but it entangles with its surroundings in a dynamic interplay. Researchers have long accounted for the complexity of these intermolecular arrangements through a rather phenomenological description. But now the focus is shifting toward a detailed understanding of supramolecular structure at atomic resolution, constantly expanding our understanding of the stunning influence of the environment. In this Account, we discuss how ssNMR can help to dissect the remarkable interplay between intra- and intermolecular interactions. We describe biochemical and spectroscopic strategies that tailor ssNMR spectroscopic methods to the challenge of supramolecular structure investigation. In particular, we consider protein-protein interactions or the protein-membrane topology, and we review recent applications of these techniques. Furthermore, we summarize methods for integrating ssNMR information with other experimental techniques or computational methods, and we offer perspectives on how this overall information allows us to target increasingly large and intricate supramolecular structures of biomolecules. Advancements in ssNMR methodology and instrumentation, including the incorporation of signal enhancement methods such as dynamic nuclear polarization will further increase the potential of ssNMR spectroscopy, and together with additional developments in the field of NMR-hybrid strategies, ssNMR may become an ideal tool to study the heterogeneous, dynamic, and often transient nature of molecular interactions in complex biological systems.

Structural Determinants of Specific Lipid Binding to Potassium Channels

We demonstrate that heteronuclear decoupling using a Phase-Inverted Supercycled Sequence for Attenuation of Rotary ResOnance (PISSARRO) is very efficient at high spinning frequencies (nu(rot)=60kHz) and high magnetic fields (900MHz for protons at 21T) even with moderate radio-frequency decoupling amplitudes (nu(1)(I)=15kHz), despite the wide range of isotropic chemical shifts of the protons and the increased effect of their chemical shift anisotropy.

Quantitative Analysis of the Water Occupancy around the Selectivity Filter of a K+ Channel in Different Gating Modes

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 channels 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.

Broadband Carbon-13 Correlation Spectra of Microcrystalline Proteins in Very High Magnetic Fields

Recovery in K(+) channels, that is, the transition from the inactivated nonconductive selectivity filter conformation toward the conductive conformation, occurs on a time scale of the order of seconds, which is astonishingly long, given that the structural differences among the filter conformations are faint (<1 Å). Computational studies and electrophysiological measurements suggested that buried water molecules bound behind the selectivity filter are at the origin of the slowness of recovery in K(+) channels. Using a combination of solid-state NMR spectroscopy (ssNMR) and long molecular dynamics simulations, we sketch a high-resolution map of the spatial and temporal distribution of water behind the selectivity filter of a membrane-embedded K(+) channel in two different gating modes. Our study demonstrates that buried water molecules with long residence times are spread all along the rear of the inactivated filter, which explains the recovery kinetics. In contrast, the same region of the structure appears to be dewetted when the selectivity filter is in the conductive state. Using proton-detected ssNMR on fully protonated channels, we demonstrate the presence of a pathway that allows for the interchange of buried and bulk water, as required for a functional influence of buried water on recovery and slow inactivation. Furthermore, we provide direct experimental evidence for the presence of additional ordered water molecules that surround the filter and that are modulated by the channels gating mode.

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

PARIS recoupling irradiation, despite a low rf amplitude, can promote efficient magnetization transfer during solid-state NMR experiments at 900 MHz over a wide range of differences in isotropic chemical shifts in microcrystalline proteins.

Solid-State NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility

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 well-characterized 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.

Structural characterization of 13C-enriched humins and alkali-treated 13C humins by 2D solid-state NMR

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamAs transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.

Revealing molecular self-assembly and geometry of non-covalent halogen bonding by solid-state NMR spectroscopy

Humin by-products are formed during the acid-catalyzed dehydration of carbohydrates to bio-based platform molecules, such as hydroxymethylfurfural and levulinic acid. The molecular structure of these humins has not yet been unequivocally established. 1D 13C solid-state NMR data reported have, for example, provided considerable insight, but do not allow for the unambiguous assignment of key structural motifs. Complementary (2D) techniques are needed to gain additional insight into the molecular structure of humins. Here, the preparation of 13C-enriched humins is reported, together with the reactive solubilization of these labeled humins and their characterization with complementary 1D and 2D solid-state NMR techniques. 1D cross polarization (CP) and direct excitation (DE) 13C solid-state NMR spectra, 2D 13C-detected double-quantum single-quantum (DQSQ) as well as 2D 1H-detected heteronuclear correlation (HETCOR) were recorded with different excitation schemes. These experiments unambiguously established that the original humins have a furan-rich structure with aliphatic linkers and allowed for a refinement of the molecular structure proposed previously. Solid-state NMR data of alkali-treated 13C-labeled humins showed that an arene-rich structure is formed at the expense of the furanic network during alkaline pretreatment.