The 3D structure of lipidic fibrils of α-synuclein.

α-synuclein misfolding and aggregation into fibrils is a common feature of α-synucleinopathies, such as Parkinson's disease, in which α-synuclein fibrils are a characteristic hallmark of neuronal inclusions called Lewy bodies. Studies on the composition of Lewy bodies extracted postmortem from brain tissue of Parkinson's patients revealed that lipids and membranous organelles are also a significant component. Interactions between α-synuclein and lipids have been previously identified as relevant for Parkinson's disease pathology, however molecular insights into their interactions have remained elusive. Here we present cryo-electron microscopy structures of six α-synuclein fibrils in complex with lipids, revealing specific lipid-fibril interactions. We observe that phospholipids promote an alternative protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities of the fibrils. Together with our previous studies, these structures also indicate a mechanism for fibril-induced lipid extraction, which is likely to be involved in the development of α-synucleinopathies. Specifically, one potential mechanism for the cellular toxicity is the disruption of intracellular vesicles mediated by fibrils and oligomers, and therefore the modulation of these interactions may provide a promising strategy for future therapeutic interventions.

Atomic resolution dynamics of cohesive interactions in phase-separated Nup98 FG domains.

Cohesive FG domains assemble into a condensed phase forming the selective permeability barrier of nuclear pore complexes. Nanoscopic insight into fundamental cohesive interactions has long been hampered by the sequence heterogeneity of native FG domains. We overcome this challenge by utilizing an engineered perfectly repetitive sequence and a combination of solution and magic angle spinning NMR spectroscopy. We map the dynamics of cohesive interactions in both phase-separated and soluble states at atomic resolution using TROSY for rotational correlation time (TRACT) measurements. We find that FG repeats exhibit nanosecond-range rotational correlation times and remain disordered in both states, although FRAP measurements show slow translation of phase-separated FG domains. NOESY measurements enable the direct detection of contacts involved in cohesive interactions. Finally, increasing salt concentration and temperature enhance phase separation and decrease local mobility of FG repeats. This lower critical solution temperature (LCST) behaviour indicates that cohesive interactions are driven by entropy.

Modest Offset Difference Internuclear Selective Transfer via Homonuclear Dipolar Coupling.

Homonuclear dipolar recoupling is routinely used for magic-angle spinning NMR-based structure determination. In fully protonated samples, only short proton-proton distances are accessible to broadband recoupling approaches because of high proton density. Selective methods allow detection of longer distances by directing polarization to a subset of spins. Here we introduce the selective pulse sequence MODIST, which recouples spins that have a modest chemical shift offset difference, and demonstrate it to selectively record correlations between amide protons. The sequence was selected for good retention of total signal, leading to up to twice the intensity for proton-proton correlations compared with other selective methods. The sequence is effective across a range of spinning conditions and magnetic fields, here tested at 55.555 and 100 kHz magic-angle spinning and at proton Larmor frequencies from 600 to 1200 MHz. For influenza A M2 in lipid bilayers, cross-peaks characteristic of a helical conformation are observed.

Structure and Gating Behavior of the Human Integral Membrane Protein VDAC1 in a Lipid Bilayer.

The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the ß-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1's 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of ß strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel's structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded ß-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein's functional structure. Using a quintuple mutant implementing the channel's closed state, we found that dynamics are a key element in the protein's gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR.

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.2 GHz proton frequency) and ultrafast MAS spinning (<100 kHz) promise to accelerate acquisition, while reducing sample requirement, both of which are critical to membrane protein studies. Here, we review recent advances in ssNMR methodology used for structure determination of membrane proteins in native and mimetic environments, as well as the study of protein functions such as protein dynamics, and interactions with ligands, lipids and cholesterol.

Structure, gating and interactions of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel's function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the ß-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol.

Correcting for magnetic field drift in magic-angle spinning NMR datasets.

Magnetic field drift during magic angle spinning (MAS) NMR measurements is detrimental to the spectra, causing broadening of lines and distortion of lineshapes, especially in high-quality samples with linewidths of less than 0.1 ppm. We report that a simple linear correction for magnet drift can be used to improve the quality of proton detected MAS NMR measurements. Despite the fact that the magnetic field of superconducting magnets changes in a non-linear fashion, we find that when data acquisition is sufficiently short, a linear correction is a good approximation to the actual field drift. We used a script written in the C programming language for linear drift correction of multidimensional datasets (2D, 3D, 4D), which can be executed directly from Bruker Topspin. A second script allows datasets to be subdivided into arbitrarily short measurements, individually corrected, and concatenated before processing.

Alpha protons as NMR probes in deuterated proteins.

We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα-Hα and Hα-HN contacts present in ß-sheets become accessible, improving the chance to determine the protein structure as compared with HN-HN contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R2 relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR.

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.

Identification of Serine Conformers by Matrix-Isolation IR Spectroscopy Aided by Near-Infrared Laser-Induced Conformational Change, 2D Correlation Analysis, and Quantum Mechanical Anharmonic Computations.

The conformers of α-serine were investigated by matrix-isolation IR spectroscopy combined with NIR laser irradiation. This method, aided by 2D correlation analysis, enabled unambiguously grouping the spectral lines to individual conformers. On the basis of comparison of at least nine experimentally observed vibrational transitions of each conformer with empirically scaled (SQM) and anharmonic (GVPT2) computed IR spectra, six conformers were identified. In addition, the presence of at least one more conformer in Ar matrix was proved, and a short-lived conformer with a half-life of (3.7 ± 0.5) × 10(3) s in N2 matrix was generated by NIR irradiation. The analysis of the NIR laser-induced conversions revealed that the excitation of the stretching overtone of both the side chain and the carboxylic OH groups can effectively promote conformational changes, but remarkably different paths were observed for the two kinds of excitations.

Near-infrared laser-induced structural changes of glycine·water complexes in an Ar matrix.

The structures of glycine·H2O complexes have been reinvestigated in low-temperature inert matrices. To go beyond the former matrix-isolation IR studies, NIR laser irradiation was used to change the relative abundances of the different complexes in the matrix. It is shown that the irradiation of the first overtone of the OH stretching mode of glycine as well as of the first overtone of the OH stretching mode of the water molecule in the complex can induce structural changes. Comparison of the experimental IR spectra with the IR spectra computed for different structures resulted in more reliable assignments of spectral patterns and identification of more structures than in former studies.

Exploring the conformational space of cysteine by matrix isolation spectroscopy combined with near-infrared laser induced conformational change.

Six conformers of α-cysteine were identified by matrix isolation IR spectroscopy combined with NIR laser irradiation. Five of these conformers are identical with the five out of six conformers that have recently been identified by microwave spectroscopy. The sixth conformer observed in the present study is a short-lived conformer, which decays by H-atom tunneling; its half-life in a 12 K N2 matrix is (1.1 ± 0.5) × 10(3) s. This study proves that matrix isolation IR spectroscopy combined with NIR laser irradiation is a suitable method to identify conformers of a complex system for which computations predict several dozens of conformers, and that the reliability of this method for conformational assignment is comparable to that of microwave spectroscopy.

Near-infrared laser induced conformational change of alanine in low-temperature matrixes and the tunneling lifetime of its conformer VI.

The near- and mid-IR spectra of α-alanine isolated in low-temperature Ar, Kr, and N2 matrixes were measured. Production of the short-lived conformer VI at the expense of the predominant conformer I was observed upon short irradiation with NIR laser light at the first O-H stretching overtone band of conformer I. Conformer VI decays by H-atom tunneling at 12 K with half-lives of 5.7 ± 1 s, 2.8 ± 1 s in Ar (two different sites), 7.0 ± 1 s in Kr, and 2.8 × 10(3) ± 1.2 × 10(3) s in N2. Upon prolonged irradiation, conformer I slowly transformed into conformer IIa. On the basis of these irradiation experiments, the unambiguous vibrational assignments of conformers I, IIa, and VI are given. In contrast to similar experiments for glycine, the irradiation experiments did not lead to the formation of conformer IIIb. This is explained by a very low IIIb → I barrier height computed for alanine, which results in a very fast depletion of conformer IIIb even in low-temperature matrixes.