Segmental Dynamics of Membranous Cholesterol are Coupled.

Cholesterol promotes the structural integrity of the fluid cell membrane and interacts dynamically with many membrane proteins to regulate function. Understanding site-resolved cholesterol structural dynamics is thus important. This long-standing challenge has thus far been addressed, in part, by selective isotopic labeling approaches. Here we present a new 3D solid-state NMR (SSNMR) experiment utilizing scalar 13C-13C polarization transfer and recoupling of the 1H-13C interactions in order to determine average dipolar couplings for all 1H-13C vectors in uniformly 13C-enriched cholesterol. The experimentally determined order parameters (OP) agree exceptionally well with molecular dynamics (MD) trajectories and reveal coupling among several conformational degrees of freedom in cholesterol molecules. Quantum chemistry shielding calculations further support this conclusion and specifically demonstrate that ring tilt and rotation are coupled to changes in tail conformation and that these coupled segmental dynamics dictate the orientation of cholesterol. These findings advance our understanding of physiologically relevant dynamics of cholesterol, and the methods that revealed them have broader potential to characterize how structural dynamics of other small molecules impact their biological functions.

Hybrid measurement of respiratory aerosol reveals a dominant coarse fraction resulting from speech that remains airborne for minutes.

Accurate measurements of the size and quantity of aerosols generated by various human activities in different environments are required for efficacious mitigation strategies and accurate modeling of respiratory disease transmission. Previous studies of speech droplets, using standard aerosol instrumentation, reported very few particles larger than 5 µm. This starkly contrasts with the abundance of such particles seen in both historical slide deposition measurements and more recent light scattering observations. We have reconciled this discrepancy by developing an alternative experimental approach that addresses complications arising from nucleated condensation. Measurements reveal that a large volume fraction of speech-generated aerosol has diameters in the 5- to 20-µm range, making them sufficiently small to remain airborne for minutes, not hours. This coarse aerosol is too large to penetrate the lower respiratory tract directly, and its relevance to disease transmission is consistent with the vast majority of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiating in the upper respiratory tract. Our measurements suggest that in the absence of symptoms such as coughing or sneezing, the importance of speech-generated aerosol in the transmission of respiratory diseases is far greater than generally recognized.

Concordance of X-ray and AlphaFold2 Models of SARS-CoV-2 Main Protease with Residual Dipolar Couplings Measured in Solution.

The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N-1HN and 13C'-1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.

Hydrating the respiratory tract: An alternative explanation why masks lower severity of COVID-19.

The seasonality of respiratory diseases has been linked, among other factors, to low outdoor absolute humidity and low indoor relative humidity, which increase evaporation of water in the mucosal lining of the respiratory tract. We demonstrate that normal breathing results in an absorption-desorption cycle inside facemasks, in which supersaturated air is absorbed by the mask fibers during expiration, followed by evaporation during inspiration of dry environmental air. For double-layered cotton masks, which have considerable heat capacity, the temperature of inspired air rises above room temperature, and the effective increase in relative humidity can exceed 100%. We propose that the recently reported, disease-attenuating effect of generic facemasks is dominated by the strong humidity increase of inspired air. This elevated humidity promotes mucociliary clearance of pathogens from the lungs, both before and after an infection of the upper respiratory tract has occurred. Effective mucociliary clearance can delay and reduce infection of the lower respiratory tract, thus mitigating disease severity. This mode of action suggests that masks can benefit the wearer even after an infection in the upper respiratory tract has occurred, complementing the traditional function of masks to limit person-to-person disease transmission. This potential therapeutical use should be studied further.

Hydrating the Respiratory Tract: An Alternative Explanation Why Masks Lower Severity of COVID-19 Disease.

Seasonality of respiratory diseases has been linked, among other factors, to low outdoor absolute humidity and low relative humidity in indoor environments, which increase evaporation of water in the mucosal layer lining the respiratory tract. We demonstrate that normal breathing results in an absorption-desorption cycle inside facemasks, where super-saturated air is absorbed by the mask fibers during expiration, followed by evaporation during inspiration of dry environmental air. For double-layered cotton masks, which have considerable heat capacity, the temperature of inspired air rises above room temperature, and the effective increase in relative humidity can exceed 100%. We propose that the recently reported, disease-attenuating effect of generic facemasks is dominated by the strong humidity increase of inspired air. SIGNIFICANCE STATEMENT: Facemasks are the most widely used tool for mitigating the spread of the COVID-19 pandemic. Decreased disease severity by the wearer has also been linked to the use of cloth facemasks. This well-documented finding is surprising considering that such masks are poor at filtering the smallest aerosol particles, which can reach the lower respiratory tract and have been associated with severe disease. We show that facemasks strongly increase the effective humidity of inhaled air, thereby promoting hydration of the respiratory epithelium which is known to be beneficial to the immune system. Increased humidity of inspired air could be an alternate explanation for the now well-established link between mask wearing and lower disease severity.

Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: a single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the 15N or 13C' chemical shifts of the unfolded protein at 1 bar to the 1H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of proteins present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate 15N shifts differ strongly from natively folded protein, its 13C' chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state.

Monitoring 15N Chemical Shifts During Protein Folding by Pressure-Jump NMR.

Pressure-jump hardware permits direct observation of protein NMR spectra during a cyclically repeated protein folding process. For a two-state folding protein, the change in resonance frequency will occur nearly instantaneously when the protein clears the transition state barrier, resulting in a monoexponential change of the ensemble-averaged chemical shift. However, protein folding pathways can be more complex and contain metastable intermediates. With a pseudo-3D NMR experiment that utilizes stroboscopic observation, we measure the ensemble-averaged chemical shifts, including those of exchange-broadened intermediates, during the folding process. Such measurements for a pressure-sensitized mutant of ubiquitin show an on-pathway kinetic intermediate whose 15N chemical shifts differ most from the natively folded protein for strands ß5, its preceding turn, and the two strands that pair with ß5 in the native structure.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell.

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-µs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 µM, oligomeric off-pathway intermediates compete with folding of the native state.

Resonance assignments of an α-synuclein fibril prepared in Tris buffer at moderate ionic strength.

Fibrils of the protein α-synuclein (α-syn) are implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We have reported a high-resolution structure (PDB 2N0A) of an α-syn fibril form prepared by in vitro incubation of monomeric protein in 50 mM sodium phosphate buffer pH 7.4 with 0.1 mM EDTA and 0.01% sodium azide. In parallel with this structure determination, ongoing studies of small molecule ligands binding to α-syn fibrils, prepared in 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer, have been in progress, and it is therefore of interest to determine the structural similarity of these forms. Here we report the 13C and 15N resonance assignments for α-syn fibrils prepared with Tris-HCl buffer (pH 7.7 at 37 °C) and 100 mM NaCl. These fibrillization conditions yield a form with fibril core chemical shifts highly similar to those we reported (BMRB 16939) in the course of determining the high-resolution 2N0A structure, with the exception of some small perturbations from T44 to V55, including two sets of peaks observed for residues T44-V48. Additional differences occur in the patterns of observed residues in the primarily unstructured N-terminus. These results demonstrate a common fold of the fibril core for α-syn fibrils prepared in phosphate or Tris-HCl buffer at moderate ionic strength.

Solid-State NMR of highly 13C-enriched cholesterol in lipid bilayers.

Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.

Efficient dipolar double quantum filtering under magic angle spinning without a (1)H decoupling field.

We present a systematic study of dipolar double quantum (DQ) filtering in (13)C-labeled organic solids over a range of magic-angle spinning rates, using the SPC-n recoupling sequence element with a range of n symmetry values from 3 to 11. We find that efficient recoupling can be achieved for values n⩾7, provided that the (13)C nutation frequency is on the order of 100kHz or greater. The decoupling-field dependence was investigated and explicit heteronuclear decoupling interference conditions identified. The major determinant of DQ filtering efficiency is the decoupling interference between (13)C and (1)H fields. For (13)C nutation frequencies greater than 75kHz, optimal performance is observed without an applied (1)H field. At spinning rates exceeding 20kHz, symmetry conditions as low as n=3 were found to perform adequately.

Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein.

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register ß-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.

Preparation of Amyloid Fibrils for Magic-Angle Spinning Solid-State NMR Spectroscopy.

Solid-state NMR spectroscopy (SSNMR) is an established and invaluable tool for the study of amyloid fibril structure with atomic-level detail. Optimization of the homogeneity and concentration of fibrils enhances the resolution and sensitivity of SSNMR spectra. Here, we present a fibrillization and fibril processing protocol, starting from purified monomeric α-synuclein, that enables the collection of high-resolution SSNMR spectra suitable for site-specific structural analysis. This protocol does not rely on any special features of α-synuclein and should be generalizable to any other amyloid protein.

Experimental Protein Structure Verification by Scoring with a Single, Unassigned NMR Spectrum.

Standard methods for de novo protein structure determination by nuclear magnetic resonance (NMR) require time-consuming data collection and interpretation efforts. Here we present a qualitatively distinct and novel approach, called Comparative, Objective Measurement of Protein Architectures by Scoring Shifts (COMPASS), which identifies the best structures from a set of structural models by numerical comparison with a single, unassigned 2D (13)C-(13)C NMR spectrum containing backbone and side-chain aliphatic signals. COMPASS does not require resonance assignments. It is particularly well suited for interpretation of magic-angle spinning solid-state NMR spectra, but also applicable to solution NMR spectra. We demonstrate COMPASS with experimental data from four proteins--GB1, ubiquitin, DsbA, and the extracellular domain of human tissue factor--and with reconstructed spectra from 11 additional proteins. For all these proteins, with molecular mass up to 25 kDa, COMPASS distinguished the correct fold, most often within 1.5 Å root-mean-square deviation of the reference structure.