Homonuclear Simplified Preservation of Equivalent Pathways Spectroscopy.

Recently developed homonuclear transverse mixing optimal control pulses (hTROP) revealed an elegant way to enhance the detected signal in multidimensional magic-angle spinning (MAS) nuclear magnetic resonance experiments. Inspired by their work, we present two homonuclear simplified preservation of equivalent pathways spectroscopy (hSPEPS) sequences for recoupling CA-CO and CA-CB dipolar couplings under fast and ultrafast MAS rates, theoretically enabling a √2 improvement in sensitivity for each indirect dimension. The efficiencies of hSPEPS are evaluated for non-deuterated samples of influenza A M2 and bacterial rhomboid protease GlpG under two different external magnetic fields (600 and 1200 MHz) and MAS rates (55 and 100 kHz). Three-dimensional (H)CA(CO)NH, (H)CO(CA)NH, and (H)CB(CA)NH spectra demonstrate the high robustness of hSPEPS elements to excite carbon-carbon correlations, especially in the (H)CB(CA)NH spectrum, where hSPEPS outperforms the J-based sequence by a factor of, on average, 2.85.

Contrasting packing modes for tubular assemblies in chlorosomes.

The largest light-harvesting antenna in nature, the chlorosome, is a heterogeneous helical BChl self-assembly that has evolved in green bacteria to harvest light for performing photosynthesis in low-light environments. Guided by NMR chemical shifts and distance constraints for Chlorobaculum tepidum wild-type chlorosomes, the two contrasting packing modes for syn-anti parallel stacks of BChl c to form polar 2D arrays, with dipole moments adding up, are explored. Layered assemblies were optimized using local orbital density functional and plane wave pseudopotential methods. The packing mode with the lowest energy contains syn-anti and anti-syn H-bonding between stacks. It can accommodate R and S epimers, and side chain variability. For this packing, a match with the available EM data on the subunit axial repeat and optical data is obtained with multiple concentric cylinders for a rolling vector with the stacks running at an angle of 21° to the cylinder axis and with the BChl dipole moments running at an angle ߠ∼ 55° to the tube axis, in accordance with optical data. A packing mode involving alternating syn and anti parallel stacks that is at variance with EM appears higher in energy. A weak cross-peak at -6 ppm in the MAS NMR with 50 kHz spinning, assigned to C-181, matches the shift of antiparallel dimers, which possibly reflects a minor impurity-type fraction in the self-assembled BChl c.

Cryo-EM structures of lipidic fibrils of amyloid-ß (1-40).

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disease characterized by the extracellular deposition of amyloid plaques. Investigation into the composition of these plaques revealed a high amount of amyloid-ß (Aß) fibrils and a high concentration of lipids, suggesting that fibril-lipid interactions may also be relevant for the pathogenesis of AD. Therefore, we grew Aß40 fibrils in the presence of lipid vesicles and determined their structure by cryo-electron microscopy (cryo-EM) to high resolution. The fold of the major polymorph is similar to the structure of brain-seeded fibrils reported previously. The majority of the lipids are bound to the fibrils, as we show by cryo-EM and NMR spectroscopy. This apparent lipid extraction from vesicles observed here in vitro provides structural insights into potentially disease-relevant fibril-lipid interactions.

Simplified Preservation of Equivalent Pathways Spectroscopy.

Inspired by the recently proposed transverse mixing optimal control pulses (TROP) approach for improving signal in multidimensional magic-angle spinning (MAS) NMR experiments, we present simplified preservation of equivalent pathways spectroscopy (SPEPS). It transfers both transverse components of magnetization that occur during indirect evolutions, theoretically enabling a √2 improvement in sensitivity for each such dimension. We compare SPEPS transfer with TROP and cross-polarization (CP) using membrane protein and fibril samples at MAS of 55 and 100 kHz. In three-dimensional (3D) (H)CANH spectra, SPEPS outperformed TROP and CP by factors of on average 1.16 and 1.69, respectively, for the membrane protein, but only a marginal improvement of 1.09 was observed for the fibril. These differences are discussed, making note of the longer transfer time used for CP, 14 ms, as compared with 2.9 and 3.6 ms for SPEPS and TROP, respectively. Using SPEPS for two transfers in the 3D (H)CANCO experiment resulted in an even larger benefit in signal intensity, with an average improvement of 1.82 as compared with CP. This results in multifold time savings, in particular considering the weaker peaks that are observed to benefit the most from SPEPS.

Acetylation discriminates disease-specific tau deposition.

Pathogenic aggregation of the protein tau is a hallmark of Alzheimer's disease and several other tauopathies. Tauopathies are characterized by the deposition of specific tau isoforms as disease-related tau filament structures. The molecular processes that determine isoform-specific deposition of tau are however enigmatic. Here we show that acetylation of tau discriminates its isoform-specific aggregation. We reveal that acetylation strongly attenuates aggregation of four-repeat tau protein, but promotes amyloid formation of three-repeat tau. We further identify acetylation of lysine 298 as a hot spot for isoform-specific tau aggregation. Solid-state NMR spectroscopy demonstrates that amyloid fibrils formed by unmodified and acetylated three-repeat tau differ in structure indicating that site-specific acetylation modulates tau structure. The results implicate acetylation as a critical regulator that guides the selective aggregation of three-repeat tau and the development of tau isoform-specific neurodegenerative diseases.

A Study of the Activity of Adamantyl Amines against Mutant Influenza A M2 Channels Identified a Polycyclic Cage Amine Triple Blocker, Explored by Molecular Dynamics Simulations and Solid-State NMR.

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus in vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus in vitro, and one compound inhibited WT, L26F, V27A M2 in vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.

Real-time tracking of drug binding to influenza A M2 reveals a high energy barrier.

The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55-100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

Great Offset Difference Internuclear Selective Transfer.

Carbon-carbon dipolar recoupling sequences are frequently used building blocks in routine magic-angle spinning NMR experiments. While broadband homonuclear first-order dipolar recoupling sequences mainly excite intra-residue correlations, selective methods can detect inter-residue transfers and long-range correlations. Here, we present the great offset difference internuclear selective transfer (GODIST) pulse sequence optimized for selective carbonyl or aliphatic recoupling at fast magic-angle spinning, here, 55 kHz. We observe a 3- to 5-fold increase in intensities compared with broadband RFDR recoupling for perdeuterated microcrystalline SH3 and for the membrane protein influenza A M2 in lipid bilayers. In 3D (H)COCO(N)H and (H)CO(CO)NH spectra, inter-residue carbonyl-carbonyl correlations up to about 5 Å are observed in uniformly 13C-labeled proteins.

Windowed cross polarization at 55 kHz magic-angle spinning.

Cross polarization (CP) transfers via Hartmann-Hahn matching conditions are one of the cornerstones of solid-state magic-angle spinning NMR experiments. Here we investigate a windowed sequence for cross polarization (wCP) at 55 kHz magic-angle spinning, placing one window (and one pulse) per rotor period on one or both rf channels. The wCP sequence is known to have additional matching conditions. We observe a striking similarity between wCP and CP transfer conditions when considering the flip angle of the pulse rather than the rf-field strength applied during the pulse. Using fictitious spin-1/2 formalism and average Hamiltonian theory, we derive an analytical approximation that matches these observed transfer conditions. We recorded data at spectrometers with different external magnetic fields up to 1200 MHz, for strong and weak heteronuclear dipolar couplings. These transfers, and even the selectivity of CP were again found to relate to flip angle (average nutation).

Covalently Functionalized Egyptian Blue Nanosheets for Near-Infrared Bioimaging.

Fluorophores emitting in the near-infrared (NIR) wavelength region present optimal characteristics for photonics and especially bioimaging. Unfortunately, only few NIR fluorescent materials are known, and even fewer are biocompatible. For this reason, the scientific interest in designing NIR fluorophores is very high. Egyptian Blue (CaCuSi4O10, EB) is an NIR fluorescent layered silicate that can be exfoliated into fluorescent nanosheets (EB-NS). So far, its surface chemistry has not been tailored, but this is crucial for colloidal stability and biological targeting. Here, we demonstrate covalent surface functionalization of EB nanosheets (EBfunc) via Si-H activation using hydrosilanes with variable functionalities. In the first part of this work, EB-NS are grafted with the visible fluorescent pyrene (Pyr) moieties to demonstrate conjugation by colocalization of the Vis/NIR fluorescence on the (single) EB-NS level. Next, the same grafting procedure was repeated and validated with carboxyl group (COOH)-containing hydrosilanes. These groups serve as a generic handle for further (bio)functionalization of the EB-NS surface. In this way, folic acid (FA) could be conjugated to EB-NS, allowing the targeting of folic acid receptor-expressing cancer cells. These results highlight the potential of this surface chemistry approach to modify EB-NS, enabling targeted NIR imaging for biomedical applications.

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.

The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils.

Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two ß-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well.

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning.

Since the first pioneering studies on small deuterated peptides dating more than 20 years ago, 1H 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 1H-1H dipolar couplings, so that a direct detection of 1H signals, for long impossible without proton dilution, has become possible at high resolution. The switch from the traditional MAS NMR approaches with 13C and 15N detection to 1H boosts the signal by more than an order of magnitude, accelerating the site-specific analysis and opening the way to more complex immobilized biological systems of higher molecular weight and available in limited amounts. This paper reviews the concepts underlying this recent leap forward in sensitivity and resolution, presents a detailed description of the experimental aspects of acquisition of multidimensional correlation spectra with fast MAS, and summarizes the most successful strategies for the assignment of the resonances and for the elucidation of protein structure and conformational dynamics. It finally outlines the many examples where 1H-detected MAS NMR has contributed to the detailed characterization of a variety of crystalline and noncrystalline biomolecular targets involved in biological processes ranging from catalysis through drug binding, viral infectivity, amyloid fibril formation, to transport across lipid membranes.

Direct nitrogen interception from chitin/chitosan for imidazo[1,5-a]pyridines.

A catalyst-free one-pot methodology that enables direct nitrogen interception of chitosan/chitin for imidazo[1,5-a]pyridines was developed. This strategy features direct synthesis of important deuterated imidazo[1,5-a]pyridines and tridentate ligands. In particular, a broad group of previously inaccessible products including saturated 1-alkylimidazo[1,5-a]pyridines are unprecedently synthesized by this protocol.

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.

Direct Detection of Bound Ammonium Ions in the Selectivity Filter of Ion Channels by Solid-State NMR.

The flow of ions across cell membranes facilitated by ion channels is an important function for all living cells. Despite the huge amount of structural data provided by crystallography, elucidating the exact interactions between the selectivity filter atoms and bound ions is challenging. Here, we detect bound 15N-labeled ammonium ions as a mimic for potassium ions in ion channels using solid-state NMR under near-native conditions. The non-selective ion channel NaK showed two ammonium peaks corresponding to its two ion binding sites, while its potassium-selective mutant NaK2K that has a signature potassium-selective selectivity filter with four ion binding sites gave rise to four ammonium peaks. Ions bound in specific ion binding sites were identified based on magnetization transfer between the ions and carbon atoms in the selectivity filters. Magnetization transfer between bound ions and water molecules revealed that only one out of four ions in the selectivity filter of NaK2K is in close contact with water, which is in agreement with the direct knock-on ion conduction mechanism where ions are conducted through the channel by means of direct interactions without water molecules in between. Interestingly, the potassium-selective ion channels investigated here (NaK2K and, additionally, KcsA-Kv1.3) showed remarkably different chemical shifts for their bound ions, despite having identical amino acid sequences and crystal structures of their selectivity filters. Molecular dynamics simulations show similar ion binding and conduction behavior between ammonium and potassium ions and identify the origin of the differences between the investigated potassium channels.

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.