Marc Baldus

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR.

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane—similar to the catalytic function of the active site of an enzyme—and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel–scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton–proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein–inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin–K+ channel interactions.

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinsons disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.

Backbone and Side-Chain 13C and 15N Signal Assignments of the α-Spectrin SH3 Domain by Magic Angle Spinning Solid-State NMR at 17.6 Tesla

The backbone and side‐chain 13C and 15N signals of a solid 62‐residue (u‐13C,15N)‐labelled protein containing the α‐spectrin SH3 domain were assigned by two‐dimensional (2D) magic angle spinning (MAS) 15N–13C and 13C–13C dipolar correlation spectroscopy at 17.6 T. The side‐chain signal sets of the individual amino acids were identified by 2D 13C–13C proton‐driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton–nitrogen and a nitrogen–carbon cross‐polarisation step followed by a carbon–carbon homonuclear transfer unit. Interresidue correlations leading to sequence‐specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7–61, while the signals of the N‐ and C‐terminal residues 1–6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor‐bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three‐dimensional versions of the applied or analogous techniques.

The conformation of neurotensin bound to its G protein-coupled receptor

G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13C,15N-labeled NT(8–13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a β-strand conformation. Our results provide a viable structural template for further pharmacological investigations.

Solid‐State NMR Spectroscopy on Complex Biomolecules

Biomolecular applications of NMR spectroscopy are often merely associated with soluble molecules or magnetic resonance imaging. However, since the late 1970s, solid-state NMR (ssNMR) spectroscopy has demonstrated its ability to provide atomic-level insight into complex biomolecular systems ranging from lipid bilayers to complex biomaterials. In the last decade, progress in the areas of NMR spectroscopy, biophysics, and molecular biology have significantly expanded the repertoire of ssNMR spectroscopy for biomolecular studies. This Review discusses current approaches and methodological challenges, and highlights recent progress in using ssNMR spectroscopy at the interface of structural and cellular biology.

Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization†

Solid-state NMR (ssNMR) spectroscopy offers increasing possibilities to study complex biomolecules at the atomic level. An important target area concerns membrane-associated proteins, which can be investigated by ssNMR methods after reconstitution in synthetic bilayers. While such preparations allow examination of functional aspects of the protein of interest, the influence of the native cellular environment on protein structure and function cannot be monitored. Very recently, we introduced a general approach aimed at determining complex molecular structures, including integral membrane proteins, in their native cellular environment by ssNMR under magic-angle-spinning (MAS) conditions. Using dedicated sample-preparation routes, we demonstrated that high-resolution ssNMR spectra can be obtained on uniformly C,N-labeled preparations of Escherichia coli whole cells (WC) and cell envelopes (CE). Both CE and WC morphology are preserved under standard ssNMR experimental conditions and the corresponding C and N crosspolarization (CP-MAS) spectra are invariant over time. However, with increasing levels of molecular complexity, especially in the case of WC preparations, spectroscopic sensitivity becomes a critical factor. In recent years, dynamic nuclear polarization (DNP) has developed into a routine tool to increase the sensitivity of multidimensional ssNMR. DNP enhancements of up to 148fold have been obtained on micro/nanocrystalline biomolecular samples, including an amyloidogenic peptide and a deuterated protein, 6] while enhancements between 18and 46fold have been reported for membrane-embedded polypeptides, purple membrane preparations, and bacteriophages. Here, we investigated the use of DNP to conduct ssNMR studies on C,N-labeled preparations of E. coli WC overproducing the integral outer membrane protein PagL. In Figure 1, we compared C and N CP-MAS spectra of uniformly C,N-labeled WC with the CE isolated from PagL-overproducing E. coli cells, recorded in the presence and absence of microwave irradiation. At higher temperatures (271 K), ssNMR spectra of the E. coli CE had previously revealed atomic details of PagL as well as endogenous membrane-associated macromolecules, including the major lipoprotein Lpp and non-proteinaceous components such as lipopolysaccharides (LPS), peptidoglycans (PG), and phospholipids. Under low-temperature (LT) DNP conditions, we observed significant DNP enhancement factors for both preparations in spectral regions characteristic for protein signals (aliphatic C resonances: d = 50–55 ppm, amide N backbone and side-chain resonances at about 120 and 80–30 ppm) as well as for C signals of endogenous

3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints

In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.

Cellular solid-state nuclear magnetic resonance spectroscopy

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

Characterization of Alzheimer's-like Paired Helical Filaments from the Core Domain of Tau Protein Using Solid-State NMR Spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimers disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.