Identification of potential aggregation hotspots on Aβ42 fibrils blocked by the anti-amyloid chaperone-like BRICHOS domain.

Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer's disease (AD). The surface of AD-associated amyloid-β peptide (Aβ) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle.

Heteronuclear decoupling with rotor-synchronized phase-alternated cycles.

A new heteronuclear decoupling pulse sequence is introduced, dubbed ROtor-Synchronized Phase-Alternated Cycles (ROSPAC). It is based on a partial refocusing of the coherences (spin operator products or cross-terms) [Filip et al., J.

H-Detected Biomolecular NMR under Fast Magic-Angle Spinning.

Since the first pioneering studies on small deuterated peptides dating more than 20 years ago, H detection has evolved into the most efficient approach for investigation of biomolecular structure, dynamics, and interactions by solid-state NMR. The development of faster and faster magic-angle spinning (MAS) rates (up to 150 kHz today) at ultrahigh magnetic fields has triggered a real revolution in the field. This new spinning regime reduces the H-H dipolar couplings, so that a direct detection of H signals, for long impossible without proton dilution, has become possible at high resolution.

Protein structural dynamics by Magic-Angle Spinning NMR.

Magic-Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR) is a fast-developing technique, capable of complementing solution NMR, X-ray crystallography, and electron microscopy for the biophysical characterization of microcrystalline, poorly crystalline or disordered protein samples, such as enzymes, biomolecular assemblies, membrane-embedded systems or fibrils. Beyond structures, MAS NMR is an ideal tool for the investigation of dynamics, since it is unique in its ability to distinguish static and dynamic disorder, and to characterize not only amplitudes but also timescales of motion. Building on seminal work on model proteins, the technique is now ripe for widespread application in structural biology.

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics.

Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms.

A β-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters.

Conformational Stability and Dynamics in Crystals Recapitulate Protein Behavior in Solution.

A growing body of evidences has established that in many cases proteins may preserve most of their function and flexibility in a crystalline environment, and several techniques are today capable to characterize molecular properties of proteins in tightly packed lattices. Intriguingly, in the case of amyloidogenic precursors, the presence of transiently populated states (hidden to conventional crystallographic studies) can be correlated to the pathological fate of the native fold; the low fold stability of the native state is a hallmark of aggregation propensity. It remains unclear, however, to which extent biophysical properties of proteins such as the presence of transient conformations or protein stability characterized in crystallo reflect the protein behavior that is more commonly studied in solution.

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR.

Most commonly small outer membrane proteins, possessing between 8 and 12 β-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded β-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules.

Protein NMR Resonance Assignment without Spectral Analysis: 5D SOlid-State Automated Projection SpectroscopY (SO-APSY).

Narrow proton signals, high sensitivity, and efficient coherence transfers provided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible for the solid-state NMR analysis of proteins. We present the first ultrahigh dimensional implementation of this approach, where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular sizes and aggregation states, which show limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis and yield rapid and unbiased assignments of backbone resonances.

Conformational dynamics in crystals reveal the molecular bases for D76N beta-2 microglobulin aggregation propensity.

Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process. A prototypic example is the D76N mutant of beta-2 microglobulin (β2m) that displays an aggressive aggregation propensity. Here we investigate the dynamics of β2m by X-ray crystallography, solid-state NMR, and molecular dynamics simulations to unveil the effects of the D76N mutation.

H magic-angle spinning NMR evolves as a powerful new tool for membrane proteins.

Building on a decade of continuous advances of the community, the recent development of very fast (60 kHz and above) magic-angle spinning (MAS) probes has revolutionised the field of solid-state NMR. This new spinning regime reduces the H-H dipolar couplings, so that direct detection of the larger magnetic moment available from H is now possible at high resolution, not only in deuterated molecules but also in fully-protonated substrates. Such capabilities allow rapid "fingerprinting" of samples with a ten-fold reduction of the required sample amounts with respect to conventional approaches, and permit extensive, robust and expeditious assignment of small-to-medium sized proteins (up to ca.

Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution.

The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring H-N dipolar-coupling as well as N R and R relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the β-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR.

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination.

Protein residue linking in a single spectrum for magic-angle spinning NMR assignment.

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample.

High-resolution proton-detected NMR of proteins at very fast MAS.

When combined with high-frequency (currently ∼60 kHz) magic-angle spinning (MAS), proton detection boosts sensitivity and increases coherence lifetimes, resulting in narrow ((1))H lines. Herein, we review methods for efficient proton detected techniques and applications in highly deuterated proteins, with an emphasis on 100% selected ((1))H site concentration for the purpose of sensitivity. We discuss the factors affecting resolution and sensitivity that have resulted in higher and higher frequency MAS.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning.

Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems.