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Communications Biology Mar 2024Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological...
Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. We propose a detailed analysis of M3-Gq coupling through examination of previously well-defined binding interfaces and neglected regions. Our findings suggest potential binding interfaces between M3 and Gq in pre-assembled and functionally active complexes. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling.
Topics: Receptors, Muscarinic; GTP-Binding Protein alpha Subunits, Gq-G11
PubMed: 38521872
DOI: 10.1038/s42003-024-06056-1 -
Journal of Animal Science Jan 2024The aim of the present study was to determine the optimal concentration of dietary protein required in transition diets for multiparous sows that enhance the farrowing...
The aim of the present study was to determine the optimal concentration of dietary protein required in transition diets for multiparous sows that enhance the farrowing process, colostrum production, and subsequent lactation performance. Forty-eight multiparous sows were allotted to one of six dietary treatments according to body weight (290 ± 3 kg) and parity (3.8 ± 0.2) from day 108 of gestation until 24 h after the onset of farrowing. The diets were isoenergetic and contained increasing concentrations of dietary protein (expressed as standardized ileal digestible [SID] Lys) and were supplied at a daily feed supply of 3.8 kg. On day 108 of gestation and days 2, 7, 14, 21, and 28 of lactation, body weight, and back fat thickness were recorded, and blood was sampled on day 108 of gestation, at the onset of farrowing, and days 3, 10, 17, and 24 of lactation from the sows for analysis of plasma metabolites. On day 115 of gestation, urine, and feces were collected for nitrogen (N) balance. The number of liveborn and stillborn piglets and time of birth were recorded and blood from every fourth piglet was sampled at birth for blood gas analysis. Piglets were weighed individually from birth until weaning, to estimate the colostrum and milk yield of the sows. Colostrum and milk samples were collected, and their compositions were determined. On days 3 and 28 of lactation, sows were injected with deuterium oxide to estimate body composition. The N utilization was maximized when the concentration of SID Lys in the transition diet was 6.06 g/kg (P < 0.01). When urinary concentrations of urea were expressed relative to creatinine, the relative concentration of urea remained low until a dietary concentration of 6.08 g SID Lys/kg, above which the relative concentration of urea increased (P < 0.01). Stillbirth rate increased linearly with increasing SID Lys concentration in the transition diet (P < 0.001), thus the concentration of SID Lys should be kept as low as possible without impairing sow performance excessively. A carry-over effect on milk yield was observed, showing that a dietary SID Lys concentration of 5.79 g/kg during transition optimized milk production at an average yield of 13.5 kg/d (P = 0.04). Increasing loss of body fat in lactation was observed with increasing SID Lys concentration in the transition diet (P = 0.03). In conclusion, the transition diet of multiparous sows should contain 5.79 g SID Lys/kg when fed 3.8 kg/d (13.0 MJ ME/kg), for a total SID Lys intake of 22 g/d.
Topics: Pregnancy; Animals; Swine; Female; Diet; Lactation; Body Weight; Dietary Proteins; Urea; Animal Feed
PubMed: 38517473
DOI: 10.1093/jas/skae082 -
Molecular Pharmaceutics Apr 2024Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating...
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.
Topics: Humans; Deuterium; Deuterium Exchange Measurement; Excipients; Granulocyte Colony-Stimulating Factor; Mass Spectrometry; Proteins
PubMed: 38516985
DOI: 10.1021/acs.molpharmaceut.3c01211 -
The Journal of Veterinary Medical... May 2024Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus...
Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.
Topics: Animals; Larva; Bees; Tyramine; Enterococcaceae; Chromatography, Liquid; Tandem Mass Spectrometry
PubMed: 38508725
DOI: 10.1292/jvms.23-0510 -
Biophysics Reviews Jun 2023Neutron scattering methods are powerful tools for the study of the structure and dynamics of lipid bilayers in length scales from sub Å to tens to hundreds nm and the... (Review)
Review
Neutron scattering methods are powerful tools for the study of the structure and dynamics of lipid bilayers in length scales from sub Å to tens to hundreds nm and the time scales from sub ps to s. These techniques also are nondestructive and, perhaps most importantly, require no additives to label samples. Because the neutron scattering intensities are very different for hydrogen- and deuterium-containing molecules, one can replace the hydrogen atoms in a molecule with deuterium to prepare on demand neutron scattering contrast without significantly altering the physical properties of the samples. Moreover, recent advances in neutron scattering techniques, membrane dynamics theories, analysis tools, and sample preparation technologies allow researchers to study various aspects of lipid bilayer dynamics. In this review, we focus on the dynamics of individual lipids and collective membrane dynamics as well as the dynamics of hydration water.
PubMed: 38504928
DOI: 10.1063/5.0144544 -
Biochimica Et Biophysica Acta. Proteins... Jul 2024Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy,...
Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.
Topics: Epitope Mapping; Protein Footprinting; SARS-CoV-2; Hydroxyl Radical; Humans; Spike Glycoprotein, Coronavirus; Protein Binding; COVID-19; Binding Sites; Antibodies, Viral; Mass Spectrometry; Protein Domains
PubMed: 38499233
DOI: 10.1016/j.bbapap.2024.141011 -
Human Vaccines & Immunotherapeutics Dec 2024Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward...
Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.
Topics: Humans; Influenza Vaccines; Influenza, Human; Neuraminidase; Vaccines, Synthetic; RNA
PubMed: 38497413
DOI: 10.1080/21645515.2024.2304393 -
Biophysics and Physicobiology 2023Protein functions associated with biological activity are precisely regulated by both tertiary structure and dynamic behavior. Thus, elucidating the high-resolution...
Protein functions associated with biological activity are precisely regulated by both tertiary structure and dynamic behavior. Thus, elucidating the high-resolution structures and quantitative information on in-solution dynamics is essential for understanding the molecular mechanisms. The main experimental approaches for determining tertiary structures include nuclear magnetic resonance (NMR), X-ray crystallography, and cryogenic electron microscopy (cryo-EM). Among these procedures, recent remarkable advances in the hardware and analytical techniques of cryo-EM have increasingly determined novel atomic structures of macromolecules, especially those with large molecular weights and complex assemblies. In addition to these experimental approaches, deep learning techniques, such as AlphaFold 2, accurately predict structures from amino acid sequences, accelerating structural biology research. Meanwhile, the quantitative analyses of the protein dynamics are conducted using experimental approaches, such as NMR and hydrogen-deuterium mass spectrometry, and computational approaches, such as molecular dynamics (MD) simulations. Although these procedures can quantitatively explore dynamic behavior at high resolution, the fundamental difficulties, such as signal crowding and high computational cost, greatly hinder their application to large and complex biological macromolecules. In recent years, machine learning techniques, especially deep learning techniques, have been actively applied to structural data to identify features that are difficult for humans to recognize from big data. Here, we review our approach to accurately estimate dynamic properties associated with local fluctuations from three-dimensional cryo-EM density data using a deep learning technique combined with MD simulations.
PubMed: 38496243
DOI: 10.2142/biophysico.bppb-v20.0022 -
Applied Radiation and Isotopes :... Jun 2024Bruce Power operates a first-of-its-kind isotope production system (IPS) that enables continuous production of Lu within Canada Deuterium Uranium (CANDU) commercial...
Bruce Power operates a first-of-its-kind isotope production system (IPS) that enables continuous production of Lu within Canada Deuterium Uranium (CANDU) commercial power reactors. Located on the reactivity mechanisms deck of Unit 7, just outside of reactor containment but in close proximity to the primary heat transport (PHT) pumps, this facility offers unique advantages for Lu production. However, employees working in this area encounter a radiation hazard which consists primarily of photoneutrons. These originate from the base of the PHT pumps and are only present when the reactor is operating. This study evaluates neutron exposure at Bruce Power's IPS by using a nested neutron spectrometer (NNS) to determine the neutron energy spectra and absolute dosimetric quantities such as the ambient dose equivalent, H*(10). The results from the NNS are then compared to surveys performed by a portable neutron rem meter (Model NP-2 by Nuclear Research Corporation), routinely used by Bruce Power staff for workplace monitoring. While the Model NP-2 generally showed consistent results across locations, a 50% dose correction factor was identified when operators were harvesting Lu from the IPS. This finding highlights an opportunity to reduce the neutron dose that is assigned to operators when producing Lu.
PubMed: 38492278
DOI: 10.1016/j.apradiso.2024.111284 -
Proceedings of the National Academy of... Mar 2024Water microdroplets (7 to 11 µm average diameter, depending on flow rate) are sprayed in a closed chamber at ambient temperature, whose relative humidity (RH) is...
Water microdroplets (7 to 11 µm average diameter, depending on flow rate) are sprayed in a closed chamber at ambient temperature, whose relative humidity (RH) is controlled. The resulting concentration of ROS (reactive oxygen species) formed in the microdroplets, measured by the amount of hydrogen peroxide (HO), is determined by nuclear magnetic resonance (NMR) and by spectrofluorimetric assays after the droplets are collected. The results are found to agree closely with one another. In addition, hydrated hydroxyl radical cations (•OH-HO) are recorded from the droplets using mass spectrometry and superoxide radical anions (•O) and hydroxyl radicals (•OH) by electron paramagnetic resonance spectroscopy. As the RH varies from 15 to 95%, the concentration of HO shows a marked rise by a factor of about 3.5 in going from 15 to 50%, then levels off. By replacing the HO of the sprayed water with deuterium oxide (DO) but keeping the gas surrounding droplets with HO, mass spectrometric analysis of the hydrated hydroxyl radical cations demonstrates that the water in the air plays a dominant role in producing HO and other ROS, which accounts for the variation with RH. As RH increases, the droplet evaporation rate decreases. These two facts help us understand why viruses in droplets both survive better at low RH values, as found in indoor air in the wintertime, and are disinfected more effectively at higher RH values, as found in indoor air in the summertime, thus explaining the recognized seasonality of airborne viral infections.
PubMed: 38489384
DOI: 10.1073/pnas.2315940121