Four-dimensional NOE-NOE spectroscopy of SARS-CoV-2 Main Protease to facilitate resonance assignment and structural analysis

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 that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the 1H–1H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonance assignments as well as for probing structural features.

Relaxation-induced dipolar exchange with recoupling (RIDER) distortions in CODEX experiments

Chemical shift anisotropy (CSA) and dipolar CODEX (Cenralband Only Detection of EXchange) experiments enable abundant quantitative information on the reorientation of the CSA and dipolar tensors to be obtained on millisecond–second timescales. At the same time, proper performance of the experiments and data analysis can often be a challenge since CODEX is prone to some interfering effects that may lead to incorrect interpretation of the experimental results. One of the most important such effects is RIDER (relaxation-induced dipolar exchange with recoupling). It appears due to the dipolar interaction of the observed X nuclei with some other nuclei, which causes an apparent decay in the mixing time dependence of the signal intensity reflecting not molecular motion, but spin flips of the adjacent nuclei. This may hamper obtaining correct values of the parameters of molecular mobility. In this contribution we consider in detail the reasons why the RIDER distortions remain even under decoupling conditions and propose measures to eliminate them. That is, we suggest (1) using an additional Z filter between the cross-polarization (CP) section and the CODEX recoupling blocks that suppresses the interfering anti-phase coherence responsible for the X-H RIDER and (2) recording only the cosine component of the CODEX signal since it is less prone to the RIDER distortions in comparison to the sine component. The experiments were conducted on rigid model substances as well as microcrystalline 2H ∕ 15N-enriched proteins (GB1 and SH3) with a partial back-exchange of labile protons. Standard CSA and dipolar CODEX experiments reveal a fast-decaying component in the mixing time dependence of 15N nuclei in proteins, which can be misinterpreted as a slow overall protein rocking motion. However, the RIDER-free experimental setup provides flat mixing time dependences, meaning that the studied proteins do not undergo global motions on the millisecond timescale.

RIDER distortions in the CODEX experiments

CSA and dipolar CODEX experiments enable obtaining abundant quantitative information on the reorientation of the CSA and dipolar tensors on the millisecond-second time scales. At the same time, proper performance of the experiments and data analysis can often be a challenge since CODEX is prone to some interfering effects that may lead to incorrect interpretation of the experimental results. One of the most important such effects is RIDER (Relaxation Induced Dipolar Exchange with Recoupling). It appears due to the dipolar interaction of the observed X-nuclei with some other nuclei, which causes an apparent decay in the mixing time dependence of the signal intensity reflecting not molecular motion but spin-flips of the adjacent nuclei. This may hamper obtaining correct values of the parameters of molecular mobility. In this contribution we consider in detail the reasons, why the RIDER distortions remain even under decoupling conditions and propose measures to eliminate them. Namely, we suggest the additional Z-filter between the cross-polarization section and the CODEX recoupling blocks, which suppresses the interfering anti-phase coherence responsible for the X-H RIDER. The experiments were conducted on rigid model substances as well as microcrystalline 2H/15N-enriched proteins (GB1 and SH3) with a partial back-exchange of labile protons. Standard CSA and dipolar CODEX experiments reveal a fast decaying component in the mixing time dependence of 15N nuclei in proteins, which can be interpreted as a slow overall protein rocking motion. However, the RIDER-free experimental setup provides flat mixing time dependencies meaning that the studied proteins do not undergo global motions on the millisecond time scale.

Deuteros 2.0: peptide-level significance testing of data from hydrogen deuterium exchange mass spectrometry

Summary Hydrogen deuterium exchange mass spectrometry (HDX-MS) is becoming increasing routine for monitoring changes in the structural dynamics of proteins. Differential HDX-MS allows comparison of protein states, such as in the absence or presence of a ligand. This can be used to attribute changes in conformation to binding events, allowing the mapping of entire conformational networks. As such, the number of necessary cross-state comparisons quickly increases as additional states are introduced to the system of study. There are currently very few software packages available that offer quick and informative comparison of HDX-MS datasets and even fewer which offer statistical analysis and advanced visualization. Following the feedback from our original software Deuteros, we present Deuteros 2.0 which has been redesigned from the ground up to fulfill a greater role in the HDX-MS analysis pipeline. Deuteros 2.0 features a repertoire of facilities for back exchange correction, data summarization, peptide-level statistical analysis and advanced data plotting features. Availability and implementation Deuteros 2.0 can be downloaded for both Windows and MacOS from https://github.com/andymlau/Deuteros_2.0 under the Apache 2.0 license.

Preparation of Isotopically Labelled Standards of Creatinine Via H/D Exchange and Their Application in Quantitative Analysis by LC-MS

Kidneys play a crucial role in maintaining metabolic homeostasis in a body. Serum creatinine concentration is a simple test used as an indicator of renal function. One of the known ways of quantifying creatinine concentration is the liquid chromatography-mass spectrometry (LC-MS) method, using an isotopically labeled analog of creatinine as an internal standard. Unfortunately, such isotope-labeled analogs are expensive and their synthesis is complex. Here we demonstrate a facile preparation of deuterated analogues of creatinine, via the H/D exchange of hydrogens located at the α-carbon (α-C) of the N-methylated amino acid part, under basic conditions. The stability of retrieved isotopologues was analyzed under both neutral or acidic conditions, and the results revealed that the introduced deuterons do not undergo back-exchange. In addition, the coelution of deuterated and non-deuterated forms under acidic and neutral conditions was observed. The prepared isotopologues were successfully applied in the quantitative LC-MS analysis of urine samples, and the results demonstrated that the presented strategy is novel and inexpensive, and that the quantification correlates with the commonly used Jaffe test method.

Modification of pillared layered clays for scavenging radiostrontium from acidic aqueous environments

A Pillared Layered Clay (PILC), code-named FREZEN, has been developed as a potential antidote for trapping radiostrontium in the gastrointestinal tract of ruminants. The ion exchange behaviour of FREZEN has been studied, with particular emphasis on Sr+2 exchange in acidic solutions. As for most sorbents and ion ex­ changers, strontium uptake by FREZEN remains sufficiently good over a pH range of 5-11, but a sharp diminution is observed at pH values below 4. Furthermore, at lower pH values, hydronium ions (H3O+) successfully compete with strontium cations already uptaken by FREZEN. An extended "back-exchange" effect, charac­terized by fast kinetics, is thus observed. In order to impede the diffusion and prevalence of Η3Ο+, a modification of the microporous space in FREZEN has been carried out via intercalation of suitable organic molecules. Work currently underway reveals that this approach is capable of improving the performance properties of the PILC. Experimental results with glycerol as the intercalated organic agen will be presented and discussed.

Late-Stage 18O Labeling of Primary Sulfonamides via a Degradation-Reconstruction Pathway

A late-stage ¹⁸O labeling approach of sulfonamides that employs the corresponding unlabeled molecule as the starting material was developed. Upon deamination of the sulfonamide, a sulfinate intermediate was isotopically enriched using eco-friendly reagents H₂¹⁸O and ¹⁵NH₃(aq) to afford a M+5 isotopologue of the parent compound. This degradation-reconstruction approach afforded isolated yields of up to 96% for the stable isotope labeled (SIL) sulfonamides, and was compatible with multiple marketed therapeutics, including celecoxib, on a gram scale. The SIL products also exhibited no ¹⁸O/¹⁶O back exchange under extreme conditions, further validating the utility of this green strategy for drug labeling for both <i>in vitro</i> and <i>in vivo</i> use. This procedure was also adapted to include pharmaceutically relevant methyl sulfones by using ¹³CH₃, affording M+5 isotopic enrichment, thereby illustrating the broad utility of this methodology.

Late-Stage 18O Labeling of Primary Sulfonamides via a Degradation-Reconstruction Pathway

A late-stage ¹⁸O labeling approach of sulfonamides that employs the corresponding unlabeled molecule as the starting material was developed. Upon deamination of the sulfonamide, a sulfinate intermediate was isotopically enriched using eco-friendly reagents H₂¹⁸O and ¹⁵NH₃(aq) to afford a M+5 isotopologue of the parent compound. This degradation-reconstruction approach afforded isolated yields of up to 96% for the stable isotope labeled (SIL) sulfonamides, and was compatible with multiple marketed therapeutics, including celecoxib, on a gram scale. The SIL products also exhibited no ¹⁸O/¹⁶O back exchange under extreme conditions, further validating the utility of this green strategy for drug labeling for both <i>in vitro</i> and <i>in vivo</i> use. This procedure was also adapted to include pharmaceutically relevant methyl sulfones by using ¹³CH₃, affording M+5 isotopic enrichment, thereby illustrating the broad utility of this methodology.

Back-exchange of deuterium in neutron crystallography: characterization by IR spectroscopy

The application of IR spectroscopy to the characterization and quality control of samples used in neutron crystallography is described. While neutron crystallography is a growing field, the limited availability of neutron beamtime means that there may be a delay between crystallogenesis and data collection. Since essentially all neutron crystallographic work is carried out using D2O-based solvent buffers, a particular concern for these experiments is the possibility of H2O back-exchange across reservoir or capillary sealants. This may limit the quality of neutron scattering length density maps and of the associated analysis. Given the expense of central facility beamtime and the effort that goes into the production of suitably sized (usually perdeuterated) crystals, a systematic method of exploiting IR spectroscopy for the analysis of back-exchange phenomena in the reservoirs used for crystal growth is valuable. Examples are given in which the characterization of D2O/H2O back-exchange in transthyretin crystals is described.