Ad Bax

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Verified

Ad verified their affiliation via an institutional email.

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• PhD
• Group Leader at National Institutes of Health

Standard solution NMR measurements use 5-mm outer diameter (OD) sample tubes that require ca 0.5 mL of solvent to minimize “end effects” on magnetic field homogeneity in the active volume of the sample. Shigemi cells reduce the solvent requirement to ca 0.29 mL. At high ionic strength, or at ultrahigh magnetic fields, smaller OD samples are needed...

Breath particles generated deep within the lung provide noninvasive access to sampling nonvolatiles in peripheral airway lining fluid. However, background contamination, their variable production among subjects, together with a huge unknown dilution when using the common breath condensate method for collection has limited their use for quantitative...

Inclusion of residual dipolar couplings (RDCs) during the early rounds of protein structure determination requires use of a floating alignment tensor or knowledge of the alignment tensor strength and rhombicity. For proteins with interdomain motion, such analysis can falsely hide the presence of domain dynamics. We demonstrate for three proteins, m...

The soft palate and back of the throat represent vulnerable early infection sites for SARS-CoV-2, influenza, streptococci, and many other pathogens. We demonstrate that snoring causes aerosolization of pharyngeal fluid that covers these surfaces, which previously has escaped detection because the inspired airstream carries the micron-sized droplets...

Modeling of airborne virus transmission and protection against it requires knowledge of the amount of biofluid emitted into the atmosphere and its viral load. Whereas viral concentrations in biofluids are readily measured by quantitative PCR, the total volume of fluids aerosolized during speaking, as measured by different researchers using various...

Residual dipolar couplings (RDCs) are NMR measurements widely used to determine structural and dynamic information in small molecules and large macromolecules. This book provides a broad view of RDCs, from basic principles to advanced applications in organic molecules and biomolecules. Exploring the newest developments in RDC measurement and analys...

The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-depende...

Amelotin is an intrinsically disordered protein (IDP) rich in Pro residues and is involved in hydroxyapatite mineralization. It rapidly oligomerizes under physiological conditions of pH and pressure but reverts to its monomeric IDP state at elevated pressure. We identified a 105-residue segment of the protein that becomes ordered upon oligomerizati...

The important role of structural dynamics in protein function is widely recognized. Thermal or B-factors and their anisotropy, seen in x-ray analysis of protein structures, report on the presence of atomic coordinate heterogeneity that can be attributed to motion. However, their quantitative evaluation in terms of protein dynamics by x-ray ensemble...

Since the emergence of the Omicron variants at the end of 2021, they quickly became the dominant variants globally. The Omicron variants may be more easily transmitted compared to the earlier Wuhan and the other variants. In this study, we aimed to elucidate mechanisms of the altered infectivity associated with the Omicron variants. We systemically...

Considerable debate has focused on whether sampling of molecular dynamics trajectories restrained by crystallographic data can be used to develop realistic ensemble models for proteins in their natural, solution state. For the SARS-CoV-2 main protease, Mpro, we evaluated agreement between solution residual dipolar couplings (RDCs) and various recen...

Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equi...

Migration to the lungs of an initial upper airway infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other respiratory pathogens can lead to pneumonia, associated with progression from mild to severe symptoms. Chemical pneumonitis or bacterial pneumonia may be caused by the ‘macroaspiration’ of large volumes of oropharyn...

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We establi...

The 68‐kDa homodimeric 3C‐like protease of SARS‐CoV‐2, Mpro (3CLpro/Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near‐complete (>97 %) backbone assignments of a C145A variant of Mpro (MproC145A) both with, and without, the N‐te...

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We establi...

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...

The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion proce...

Due to similar reactivity of organic hydroperoxides (OHPs), an HPLC separation step is typically required for their indirect (chemical) quantification in mixtures. The high sensitivity of chemical shifts to chemical structure makes NMR an ideal tool for the simultaneous quantification of OHPs in mixtures, but the concentration of these analytes in...

Hydrogen peroxide (H 2 O 2 ) plays a key role in environmental chemistry, biology, and medicine. H 2 O 2 concentrations typically are 6 to 10 orders of magnitude lower than that of water, making its quantitative detection challenging. We demonstrate that optimized NMR spectroscopy allows direct, interference-free, quantitative measurements of H 2 O...

Although the concepts of nonuniform sampling (NUS ) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago , it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether t...

The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weakness...

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 usi...

Entry of SARS-CoV-2 into a host cell is mediated by spike, a class I viral fusion protein responsible for merging the viral and host cell membranes. Recent studies have revealed atomic-resolution models for both the postfusion 6-helix bundle (6HB) and the prefusion state of spike. However, a mechanistic understanding of the molecular basis for the...

Quantification of H2O2 concentration in aqueous solutions is of interest in many fields. It usually is based on indirect methods that rely on oxidation reactions that turn on/off fluorescent probes. Such methods can suffer from reaction incompleteness and interfering chemical species. We describe optimization of NMR detection that enables direct qu...

Dry air alters salt and water balance in the upper airways and increases the risks of COVID-19 among other respiratory diseases. We explored whether such upper airway variations in salt and water balance might alter respiratory droplet generation and potentially contribute to observed impacts of airway hydration on respiratory disease. In a randomi...

Although the concepts of non-uniform sampling (NUS) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge four decades ago (Bodenhausen and Ernst, 1981; Barna and Laue, 1987), it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data coll...

No abstract available for this article.

The SARS-CoV-2 virus is highly contagious, as demonstrated by numerous well-documented superspreading events. The infection commonly starts in the upper respiratory tract (URT) but can migrate to the lower respiratory tract (LRT) and other organs, often with severe consequences. Whereas LRT infection can lead to shedding of virus via breath and cou...

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, mak...

Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and 1H back-exchange of the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2 × 306 residues. We demonstrate th...

Resonance assignment and structural studies of larger proteins by NMR can be challenging when exchange broadening, multiple stable conformations, and back-exchanging the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2×306 residues. We demonstrate that the combination of four-dimensio...

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, where super-saturated air is...

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 sup...

Chemical denaturation is a well-established approach for probing the equilibrium between folded and unfolded states of proteins. We demonstrate applicability of this method to the detection of a small population of a transiently folded structural element in a system that is often considered to be intrinsically fully disordered. The ¹HN, ¹⁵N, ¹³Cα,...

The membrane proximal external region (MPER) of HIV‐1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660–674) exists in a monomer‐trimer equilibrium with...

HIV‐1 gp41 undergoes large structural rearrangements to mediate membrane fusion. Its membrane‐proximal external region (MPER) segment, critical to soliciting neutralizing antibodies, is shown to spontaneously assemble into an α‐helical trimer in aqueous solution. Abstract The membrane proximal external region (MPER) of HIV‐1 gp41 contains epitopes...

Droplets of oral fluid emitted by speaking are a long-recognized mechanism of respiratory virus transmission. While there have been many simulations of droplet evaporation to determine whether droplets containing virions remain floating in air or rapidly fall to the ground, they typically conceal the fundamental mechanisms because of the use of num...

Speech droplets generated by asymptomatic carriers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly considered to be a likely mode of disease transmission. Highly sensitive laser light scattering observations have revealed that loud speech can emit thousands of oral fluid droplets per second. In a closed, stagnant ai...

Speaking may be a primary mode of transmission of SARS-CoV-2. Considering that reports of asymptomatic transmission account for 50-80% of COVID-19 cases and that saliva has peak viral loads at time of patient presentation, droplet emission while speaking could be a significant factor driving transmission and warrants further study. We used a planar...

Small heat-shock proteins (sHSPs) are molecular chaperones that respond to cellular stresses to combat protein aggregation. HSP27 is a critical human sHSP that forms large, dynamic oligomers whose quaternary structures and chaperone activities depend on environmental factors. Upon exposure to cellular stresses such as heat shock or acidosis, HSP27...

Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure...

Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates the exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigat...

In aqueous solution, many biochemical reaction pathways involve reaction of an aldehyde with an amine, which progresses through generally unstable, hydrated and dehydrated, Schiff base intermediates that often are unobservable by conventional NMR. There are 4 states in the relevant equilibrium: 1) gem‐diol, 2) aldehyde, 3) hemiaminal, and 4) Schiff...

Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter case, time-ordering of the NUS...

Kurzer Prozess: Eine modifizierte CEST‐NMR‐Spektroskopiemethode ermöglichte die Detektion äußerst kurzlebiger, sehr gering populierter Schiff‐Basen‐Intermediate, die mit herkömmlicher NMR‐Spektroskopie nicht zu beobachten, aber entscheidend für die Produktbildung sind. Abstract In aqueous solution, many biochemical reaction pathways involve reacti...

Brain tissue of Alzheimer’s disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Aβ40 and Aβ42. However, it remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sa...

Over the past approximately three decades, isotope-directed NMR spectroscopy has become a powerful method for determining 3D structures of biological macromolecules and their complexes in solution. From a structural perspective NMR provides an invaluable tool for studying systems that are not amenable to crystallization, including intrinsically dis...

Although the α-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a protein’s structure. Highly charged single α-helical (SAH) domains, consisting of a high percentage (>75%) of Arg, Lys, and Glu residues, are exceptions to this rule but...

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are high...

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 condition...

Antigen presentation is a cellular process that involves a number of steps, beginning with the production of peptides by proteolysis or aberrant synthesis and the delivery of peptides to cellular compartments where they are loaded on MHC class I (MHC-I) or MHC class II (MHC-II) molecules. The selective loading and editing of high-affinity immunodom...

Novel pressure-jump NMR hardware permits direct observation of protein NMR spectra during a cyclically repeated protein folding process. While protein folding transpires, nuclei change their resonance frequencies from those of the fully disordered protein to those of the folded protein. For a two-state folding protein, the change in resonance frequ...

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain, and provide the first structural characterization of a small heat-shock protein monomer. While HSP27...

NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. R...

Significance Development of specialized instrumentation enables rapid switching of the hydrostatic pressure inside an operating NMR spectrometer. This technology allows observation of protein signals during the repeated folding process. Applied to ubiquitin, a previously extensively studied model of protein folding, the methodology reveals an initi...

3,4-dihydroxyphenylacetaldehyde (DOPAL) is a toxic and reactive product of dopamine catabolism. In the catecholaldehyde hypothesis for Parkinson's disease, it is a critical driver of the selective loss of dopaminergic neurons that characterizes the disease. DOPAL also crosslinks α-synuclein, the main component of Lewy bodies, which are a pathologic...

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and post-fusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C3 symmetr...

In unfolded proteins, peptide bonds involving Pro residues exist in an equilibrium between the minor cis and major trans conformations. Folded proteins predominantly contain trans-Pro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be a rate limiting step in protein folding. Moreover, kinases and phosphatases tha...

Using fine-tuned hydrogen bonding criteria, a library of coiled peptide fragments has been generated from a large set of high-resolution protein X-ray structures. This library is shown to be an improved representation of ϕ/ψ torsion angles seen in intrinsically disordered proteins (IDPs). The ϕ/ψ torsion angle distribution of the library, on averag...

A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a ubiquitin variant that contains two Val to A...

Background: Alpha-synuclein (αS) is a nerve cell protein associated with Parkinson disease (PD). Accumulation of αS within the enteric nervous system (ENS) and its traffic from the gut to the brain are implicated in the pathogenesis and progression of PD. αS has no known function in humans and the reason for its accumulation within the ENS is unkn...

Implementation of a new algorithm, SMILE, is described for reconstruction of non-uniformly sampled two-, three- and four-dimensional NMR data, which takes advantage of the known phases of the NMR spectrum and the exponential decay of underlying time domain signals. The method is very robust with respect to the chosen sampling protocol and, in its d...

The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd...

Supplementary Tables and Supplementary Figures

The molecular mechanism through which the interaction of a clonotypic αβ TCR with peptide/MHC (p/MHC) complexes leads to T cell activation is not yet fully understood. Here we exploit a high affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd) by combining crystallographic, NMR, and func...

Parkinson's disease has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis proposes that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxici...

Significance Parkinson’s disease is characterized by the presence in brain tissues of aberrant aggregates primarily formed by the protein α-synuclein. It has been difficult, however, to identify compounds capable of preventing the formation of such deposits because of the complexity of the aggregation process of α-synuclein. By exploiting recently...

Parkinson’s etiology has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis suggests that DOPAL, an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxicity and potent ability to oligomer...

The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optim...

Various gp41 constructs used in this study and their designations. See Fig 1 for Env precursor numbering and S2 Fig for the exact sequence of each construct. Molar masses are listed in Table 1 and S2 Fig. (PDF)

Sedimentation velocity absorbance c(s) distributions for 17–172 at pH 6 and 7 at 36°C in the presence of excess of DPC micelles. Samples were prepared as described when carrying out the SV analysis at 20°C (see Fig 2D). For details, see Materials and Methods (PDF)

Binding isotherm for the interaction of 4E10 or 2F5 with 17–172 at 28°C. (A and B) The peaks indicate the heat released after each addition of 17–172 into the antibody solution both maintained in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl and 2 mM DPC. (C) The data for titration of 2F5 with 17–172 were best fit using a single binding constant to calculate...