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.

Mobility of sodium ions in agarose gels probed through combined single- and triple-quantum NMR.

Metal ions, including biologically prevalent sodium ions, can modulate electrostatic interactions frequently involved in the stability of condensed compartments in cells. Quantitative characterization of heterogeneous ion dynamics inside biomolecular condensates demands new experimental approaches. Here we develop a 23Na NMR relaxation-based integrative approach to probe dynamics of sodium ions inside agarose gels as a model system. We exploit the electric quadrupole moment of spin-3/2 23Na nuclei and, through combination of single-quantum and triple-quantum-filtered 23Na NMR relaxation methods, disentangle the relaxation contribution of different populations of sodium ions inside gels. Three populations of sodium ions are identified: a population with bi-exponential relaxation representing ions within the slow motion regime and two populations with mono-exponential relaxation but at different rates. Our study demonstrates the dynamical heterogeneity of sodium ions inside agarose gels and presents a new experimental approach for monitoring dynamics of sodium and other spin-3/2 ions (e.g. chloride) in condensed environments.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

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.

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).

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.

Dynamically Tunable All-Weather Daytime Cellulose Aerogel Radiative Supercooler for Energy-Saving Building.

A passive cooling strategy without any electricity input has shown a significant impact on overall energy consumption globally. However, designing tunable daytime radiative cooler to meet requirement of different weather conditions is still a big challenge, especially in hot, humid regions. Here, a novel type of tunable, thermally insulating and compressible cellulose nanocrystal (CNC) aerogel coolers is prepared via chemical cross-linking and unidirectional freeze casting process. Such aerogel coolers can achieve a subambient temperature drop of 9.2 °C under direct sunlight and promisingly reached the reduction of ∼7.4 °C even in hot, moist, and fickle extreme surroundings. The tunable cooling performance can be realized via controlling the compression ratio of shape-malleable aerogel coolers. Furthermore, energy consumption modeling of using such aerogel coolers in buildings in China shows 35.4% reduction of cooling energy. This work can pave the way toward designing high-performance, thermal-regulating materials for energy consumption savings.

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.

Backbone Torsion Angle Determination Using Proton Detected Magic-Angle Spinning Nuclear Magnetic Resonance.

Protein torsion angles define the backbone secondary structure of proteins. Magic-angle spinning (MAS) NMR methods using carbon detection have been developed to measure torsion angles by determining the relative orientation between two anisotropic interactions─dipolar coupling or chemical shift anisotropy. Here we report a new proton-detection based method to determine the backbone torsion angle by recoupling NH and CH dipolar couplings within the HCANH pulse sequence, for protonated or partly deuterated samples. We demonstrate the efficiency and precision of the method with microcrystalline chicken α spectrin SH3 protein and the influenza A matrix 2 (M2) membrane protein, using 55 or 90 kHz MAS. For M2, pseudo-4D data detect a turn between transmembrane and amphipathic helices.

Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes.

Amphotericin B (AmB) is a powerful but toxic fungicide that operates via enigmatic small molecule-small molecule interactions. This mechanism has challenged the frontiers of structural biology for half a century. We recently showed AmB primarily forms extramembranous aggregates that kill yeast by extracting ergosterol from membranes. Here, we report key structural features of these antifungal 'sponges' illuminated by high-resolution magic-angle spinning solid-state NMR, in concert with simulated annealing and molecular dynamics computations. The minimal unit of assembly is an asymmetric head-to-tail homodimer: one molecule adopts an all-trans C1-C13 motif, the other a C6-C7-gauche conformation. These homodimers are staggered in a clathrate-like lattice with large void volumes similar to the size of sterols. These results illuminate the atomistic interactions that underlie fungicidal assemblies of AmB and suggest this natural product may form biologically active clathrates that host sterol guests.

Proton Detected Solid-State NMR of Membrane Proteins at 28 Tesla (1.2 GHz) and 100 kHz Magic-Angle Spinning.

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1200 MHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.

Transferred-Rotational-Echo Double Resonance.

Internuclear distance determination is the foundation for NMR-based structure calculation. However, high-precision distance measurement is a laborious process requiring lengthy data acquisitions due to the large set of multidimensional spectra needed at different mixing times. This prevents application to large or challenging molecular systems. Here, we present a new approach, transferred-rotational-echo double resonance (TREDOR), a heteronuclear transfer method in which we simultaneously detect both starting and transferred signals in a single spectrum. This co-acquisition is used to compensate for coherence decay, resulting in accurate and precise distance determination by a single parameter fit using a single spectrum recorded at an ideal mixing time. We showcase TREDOR with the microcrystalline SH3 protein using 3D spectra to resolve resonances. By combining the measured N-C and H-C distances, we calculate the structure of SH3, which converges to the correct fold, with a root-mean-square deviation of 2.1 Å compared to a reference X-ray structure. The TREDOR data used in the structure calculation were acquired in only 4 days on a 600 MHz instrument. This is achieved due to the more than 2-fold time saving afforded by co-acquisition of additional information and demonstrates TREDOR as a fast and straightforward method for determining structures via magic-angle spinning NMR.

Heteronuclear and homonuclear radio-frequency-driven recoupling.

The radio-frequency-driven recoupling (RFDR) pulse sequence is used in magic-angle spinning (MAS) NMR to recouple homonuclear dipolar interactions. Here we show simultaneous recoupling of both the heteronuclear and homonuclear dipolar interactions by applying RFDR pulses on two channels. We demonstrate the method, called HETeronuclear RFDR (HET-RFDR), on microcrystalline SH3 samples at 10 and 55.555 kHz MAS. Numerical simulations of both HET-RFDR and standard RFDR sequences allow for better understanding of the influence of offsets and paths of magnetization transfers for both HET-RFDR and RFDR experiments, as well as the crucial role of XY phase cycling.

A modification of γ-encoded RN symmetry pulses for increasing the scaling factor and more accurate measurements of the strong heteronuclear dipolar couplings.

Symmetry based γ-encoded RNnν elements are broadly used in magic-angle spinning solid-state NMR experiments to achieve selective recoupling of the heteronuclear dipolar interactions. The recoupled dipolar couplings in such experiments are scaled by a factor, Ksc, which theoretically depends on the chosen symmetry numbers N, n, and ν. However, the maximum theoretical value of Ksc for γ-encoded RNnν pulses is limited to ~0.25, resulting in long RNnν experiment times. Also, the dependence of Ksc on the experimental parameters can result in systematic errors in the experimental determination of the dipolar couplings, especially at low and moderate MAS rates. In this manuscript, we investigate the use of MODifiEd RNnν symmetry (MODERNnν(ϕM)) pulses that increase the dipolar scaling factor by at least 1.45 fold compared to γ-encoded RNnν. The second advantage of MODERNnν(ϕM) pulses with respect to traditional RNnν pulses is the reduced influence of experimental parameters on Ksc, which allows for more accurate measurement of short-range distances. The robustness of MODERNnν(ϕM) is compared with γ-encoded R1423 symmetry pulses. The enhanced performance is demonstrated on two uniformly-13C-enriched samples, N-acetyl valine and the microcrystalline protein GB1, at a 31.111 kHz MAS rate.

Exact distance measurements for structure and dynamics in solid proteins by fast-magic-angle-spinning NMR.

Fast-magic-angle-spinning solid-state NMR is a developing technique for determination of protein structure and dynamics. Proton-proton correlations usually lead to rough distance restraints, a serious hurdle towards high-resolution structures. Analogous to the "eNOE" concept in solution, an integrative approach for more accurate restraints enables improved structural accuracy with minimal analytical effort.

New experimental observations of the behavior of sodium ions in saturated rock samples.

1H NMR relaxometry of saturated rock samples has become a useful tool for the characterization of porosity and transport phenomena of enclosed fluids. The pore size can be measured using the difference between inverse relaxation values of protons absorbed by the saturated rock and that present in the bulk fluids. These experiments are usually performed at low magnetic fields to reduce the influence of the diffusion on the relaxation values in the presence of Internal Gradient Fields. Recently, sodium ions have become objects of investigation. The main advantage of sodium ions over protons as measured spins in the Petrophysic NMR experiments is their presence in water and not other phases like oil and gas. However unlike protons, sodium ions can have slow motion properties like appearance of the bi-exponential relaxation and residual quadrupolar distribution, which can lead to complex behavior of spins inside the pores. Here, we describe eight 23Na NMR experiments at 9.39 T external magnetic field, in which we have investigated the behavior of sodium ions in 4 saturated rock samples: Berea 500, Fontainebleau 1, Bentheimer sandstones, and Austin chalk. We show that the reduction of spin-spin relaxation is caused by anisotropic motion and not diffusion in the presence of Internal Gradient Fields. There can be a link between free diffusional and motional averaging regimes regardless of the size of environment in which the measured spin ions are. Using different NMR sequences, we reveal and quantitatively describe bi-exponentially relaxing spins and spins with residual quadrupolar coupling. This work demonstrates unique model for the behavior of ions inside porous media, which is different than known models (Brownstein-Tarr model and "agarose gel model").

Low-power suppression of fast-motion spin 3/2 signals.

Triple Quantum Filters (TQFs) are frequently used for the selection of bi-exponentially relaxing spin 3/2 nuclei (in particular 23Na) in ordered environments, such as biological tissues. These methods provide an excellent selection of slow-motion spins, but their sensitivity is generally low, and coherence selection requirements may lead to long experiments when applied in vivo. Alternative methods, such as 2P DIM, have demonstrated that the sensitivities of the signals from bi-exponentially relaxing sodium can be significantly increased using strategies other than TQFs. A shortcoming of the 2P DIM method is its strong dependence on B0 inhomogeneities. We describe here a method, which is sensitive to the slow-motion regime, while the signal from spins in the fast-motion regime is suppressed. This method is shown to be more effective than TQFs, requires minimal phase cycling for the suppression of the influence of rf inhomogeneity, and has less dependence on resonance offsets and B0-inhomogeneity than 2P DIM.

Site-Resolved Backbone and Side-Chain Intermediate Dynamics in a Carbohydrate-Binding Module Protein Studied by Magic-Angle Spinning NMR Spectroscopy.

Magic-angle spinning solid-state NMR spectroscopy has been applied to study the dynamics of CBM3b-Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146-residue protein has a nine-stranded ß-sandwich fold, in which 35 % of the residues are in the ß-sheet and the remainder are composed of loops and turns. Dynamically averaged (1) H-(13) C dipolar coupling order parameters were extracted in a site-specific manner by using a pseudo-three-dimensional constant-time recoupled separated-local-field experiment (dipolar-chemical shift correlation experiment; DIPSHIFT). The backbone-Cα and Cß order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water-mediated contacts, whereas other crystal contacts remain rigid.