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Solution NMR spectroscopy is a particularly powerful technique for characterizing the functional dynamics of biomolecules, which is typically achieved through the quantitative characterization of chemical exchange processes via the measurement of spin relaxation rates. In addition to the conventional nuclei such as 15 N and 13 C, which are abundant in biomolecules, fluorine-19 ( 19 F) has recently garnered attention and is being widely used as a site-specific spin probe. While 19 F offers the advantages of high sensitivity and low background, it can be susceptible to artifacts in quantitative relaxation analyses due to a multitude of dipolar and scalar coupling interactions with nearby 1 H spins. In this study, we focused on the ribose 2'- 19 F spin probe in nucleic acids and investigated the effects of 1 H- 19 F spin interactions on the quantitative characterization of slow exchange processes on the millisecond time scale. We demonstrated that the 1 H- 19 F dipolar coupling can significantly affect the interpretation of 19 F chemical exchange saturation transfer (CEST) experiments when 1 H decoupling is applied, while the 1 H- 19 F interactions have a lesser impact on Carr-Purcell-Meiboom-Gill relaxation dispersion applications. We also proposed a modified CEST scheme to alleviate these artifacts along with experimental verifications on self-complementary RNA systems. The theoretical framework presented in this study can be widely applied to various 19 F spin systems where 1 H- 19 F interactions are operative, further expanding the utility of 19 F relaxation-based NMR experiments., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes., (© 2024. The Author(s).)

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

Electrostatic interactions can play important roles in regulating various biological processes. Quantifying surface electrostatics of biomolecules is, therefore, of significant interest. Recent advances in solution NMR spectroscopy have enabled site-specific measurements of de novo near-surface electrostatic potentials (ϕ ENS ) based on a comparison of solvent paramagnetic relaxation enhancements generated from differently charged paramagnetic co-solutes with similar structures. Although the NMR-derived near-surface electrostatic potentials have been shown to agree with theoretical calculations in the context of folded proteins and nucleic acids, such benchmark comparisons may not always be possible, particularly in cases where high-resolution structural models are lacking, such as in the study of intrinsically disordered proteins. Cross-validation of ϕ ENS potentials can be achieved by comparing values obtained using three pairs of paramagnetic co-solutes, each with a different net charge. Notably we have found cases where agreement of ϕ ENS potentials between the three pairs is poor and herein we investigate the source of this discrepancy in some detail. We show that for the systems considered here ϕ ENS potentials obtained from cationic and anionic co-solutes are accurate and that the use of paramagnetic co-solutes with different structures can be a viable option for validation, although the optimal choice of paramagnetic compounds depends on the system of interest., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The measurement of spin relaxation rates provides a unique avenue for quantifying dynamic processes in biomolecules. In order to simplify analysis of the measurements so that a few key intuitive parameters can be extracted, it is often the case that experiments are designed to eliminate interference effects between different classes of spin relaxation. One example emerges in the measurement of amide proton ( 1 H N ) transverse relaxation rates in 15 N labeled proteins, where 15 N inversion pulses are applied during a relaxation element to eliminate cross-correlated spin relaxation between 1 H N - 15 N dipole- 1 H N CSA interactions. We show that unless these pulses are essentially perfect, significant oscillations in magnetization decay profiles can be obtained, due to the excitation of multiple-quantum coherences, leading potentially to errors in measured R 2 rates. With the recent development of experiments for quantifying electrostatic potentials via amide proton relaxation rates, the need for highly accurate measurement schemes becomes critical. Straightforward modifications to existing pulse sequences are suggested to achieve this goal., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Polyphenol-rich rabbiteye blueberry ( Vaccinium virgatum Aiton) leaves have attracted attention as a food material. In this study, we compared the total polyphenols, total proanthocyanidin content, and antioxidant activity of the leaves of 18 blueberry varieties and investigated the seasonal variation in polyphenols. We also evaluated the anti-cancer cell proliferation properties of the rabbiteye blueberry leaf specific cultivar 'Kunisato 35 Gou'. Rabbiteye blueberry leaves had significantly higher total polyphenol and total proanthocyanidin values than northern highbush blueberry and southern highbush blueberry leaves. The antioxidant activity of blueberry leaves was highly positively correlated with both the total polyphenol and total proanthocyanidin content. Variations were observed in the total polyphenol and total proanthocyanidin content of rabbiteye blueberry leaves harvested at different points in the growing season; leaves collected in fall to winter contained more epicatechin in addition to proanthocyanidins. In the evaluation of anti-cancer cell proliferation properties against HL-60 promyelocytic leukemia cells, the September-harvested extracts of rabbiteye blueberry 'Kunisato 35 Gou' showed strong properties, and the use of an FITC Annexin V apoptosis detection kit with propidium iodide confirmed that this HL-60 cell death occurred via apoptosis. Limiting the harvest time would make rabbiteye blueberry leaves a more functional food ingredient.

The HMQC pulse sequence and variants thereof have been exploited in studies of high molecular weight protein complexes, taking advantage of the fact that fast and slow relaxing magnetization components are sequestered along two distinct magnetization transfer pathways. Despite the simplicity of the HMQC scheme an even shorter version can be designed, based on elimination of the terminal refocusing period, as a further means of increasing signal. Here we present such an experiment, and show that significant sensitivity gains, in some cases by factors of two or more, are realized in studies of proteins varying in molecular masses from 100 kDa to 1 MDa., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: L.E.K is an associate editor of this journal (JMR)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl). Using solution NMR spectroscopy, we measured residue-specific near-surface electrostatic potentials (ϕ ENS ) of CAPRIN1 along its NaCl-induced phase separation trajectory to compare with those obtained using ATP. In both cases, electrostatic shielding decreases ϕ ENS values, yet surface potentials of CAPRIN1 in the two condensates can be different, depending on the amount of NaCl or ATP added. Our results establish that even small differences in ϕ ENS can significantly affect the level of protein enrichment and the mechanical properties of the condensed phase, leading, potentially, to the regulation of biological processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Electrostatic interactions and charge balance are important for the formation of biomolecular condensates involving proteins and nucleic acids. However, a detailed, atomistic picture of the charge distribution around proteins during the phase-separation process is lacking. Here, we use solution NMR spectroscopy to measure residue-specific near-surface electrostatic potentials ( ϕ ENS ) of the positively charged carboxyl-terminal intrinsically disordered 103 residues of CAPRIN1, an RNA-binding protein localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation. Measured ϕ ENS values have been mapped along the adenosine triphosphate (ATP)-induced phase-separation trajectory. In the absence of ATP, ϕ ENS values for the mixed state of CAPRIN1 are positive and large and progressively decrease as ATP is added. This is coupled to increasing interchain interactions, particularly between aromatic-rich and arginine-rich regions of the protein. Upon phase separation, CAPRIN1 molecules in the condensed phase are neutral ( ϕ ENS [Formula: see text] 0 mV), with ∼five molecules of ATP associated with each CAPRIN1 chain. Increasing the ATP concentration further inverts the CAPRIN1 electrostatic potential, so that molecules become negatively charged, especially in aromatic-rich regions, leading to re-entrance into a mixed phase. Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates.

It has recently been demonstrated that accurate near surface electrostatic potentials can be calculated for proteins from solvent paramagnetic relaxation enhancements (PREs) of amide protons measured using spin labels of similar structures but different charges (Yu et al. in Proc Natl Acad Sci 118(25):e2104020118, 2021). Here we develop methodology for extending such measurements to intrinsically disordered proteins at neutral pH where amide spectra are of very poor quality. Under these conditions it is shown that accurate PRE values can be measured using the haCONHA experiment that has been modified for recording 1 H α transverse relaxation rates. The optimal pulse scheme includes a spin-lock relaxation element for suppression of homonuclear scalar coupled evolution for all 1 H α protons, except those derived from Ser and Thr residues, and minimizes the radiation damping field from water magnetization that would otherwise increase measured relaxation rates. The robustness of the experiment is verified by developing a second approach using a band selective adiabatic decoupling scheme for suppression of scalar coupling modulations during 1 H α relaxation and showing that the measured PRE values from the two methods are in excellent agreement. The near surface electrostatic potential of a 103-residue construct comprising the C-terminal intrinsically disordered region of the RNA-binding protein CAPRIN1 is obtained at pH 5.5 using both 1 H N and 1 H α -based relaxation rates, and at pH 7.4 where only 1 H α rates can be quantified, with very good agreement between potentials obtained under all experimental conditions., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The human high-temperature requirement A2 (HtrA2) protein is a trimeric protease that cleaves misfolded proteins to protect cells from stresses caused by toxic, proteinaceous aggregates, and the aberrant function of HtrA2 is closely related to the onset of neurodegenerative disorders. Our methyl-transverse relaxation optimized spectroscopy (TROSY)–based NMR studies using small-peptide ligands have previously revealed a stepwise activation mechanism involving multiple distinct conformational states. However, very little is known about how HtrA2 binds to protein substrates and if the distinct conformational states observed in previous peptide studies might be involved in the processing of protein clients. Herein, we use solution-based NMR spectroscopy to investigate the interaction between the N-terminal Src homology 3 domain from downstream of receptor kinase (drk) with an added C-terminal HtrA2-binding motif (drkN SH3-PDZbm) that exhibits marginal folding stability and serves as a mimic of a physiological protein substrate. We show that drkN SH3-PDZbm binds to HtrA2 via a two-pronged interaction, involving both its C-terminal PDZ-domain binding motif and a central hydrophobic region, with binding occurring preferentially via an unfolded ensemble of substrate molecules. Multivalent interactions between several clients and a single HtrA2 trimer significantly stimulate the catalytic activity of HtrA2, suggesting that binding avidity plays an important role in regulating substrate processing. Our results provide a thermodynamic, kinetic, and structural description of the interaction of HtrA2 with protein substrates and highlight the importance of a trimeric architecture for function as a stress-protective protease that mitigates aggregation.

Developments in solution NMR spectroscopy have significantly impacted the biological questions that can now be addressed by this methodology. By means of illustration, we present here a perspective focusing on studies of a number of molecular machines that are critical for cellular homeostasis. The role of NMR in elucidating the structural dynamics of these important molecules is emphasized, focusing specifically on intersubunit allosteric communication in homo-oligomers. In many biophysical studies of oligomers, allostery is inferred by showing that models specifically including intersubunit communication best fit the data of interest. Ideally, however, experimental studies focusing on one subunit of a multisubunit system would be performed as an important complement to the more traditional bulk measurements in which signals from all components are measured simultaneously. Using an approach whereby asymmetric molecules are prepared in concert with NMR experiments focusing on the structural dynamics of individual protomers, we present examples of how intersubunit allostery can be directly observed in high-molecular-weight protein systems. These examples highlight some of the unique roles of solution NMR spectroscopy in studies of complex biomolecules and emphasize the important synergy between NMR and other atomic resolution biophysical methods., Competing Interests: The authors declare no competing interest.

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s -1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 μs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES., Competing Interests: The authors declare no competing interest.

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy-based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding-incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson's disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed., Competing Interests: The authors declare no competing interest.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults., Competing Interests: The authors declare no competing interest.

A primary biological function of multi-spanning membrane proteins is to transfer information and/or materials through a membrane by changing their conformations. Therefore, particular dynamics of the membrane proteins are tightly associated with their function. The semi-atomic resolution dynamics information revealed by NMR is able to discriminate function-related dynamics from random fluctuations. This review will discuss several studies in which quantitative dynamics information by solution NMR has contributed to revealing the structural basis of the function of multi-spanning membrane proteins, such as ion channels, GPCRs, and transporters.

The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. While computational and theoretical approaches have provided significant insights into IDR phase behavior, establishing the critical interactions that govern condensation with atomic resolution through experiment is more difficult, given the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states. NMR can be a valuable method, but the dynamic and viscous nature of condensed IDRs presents challenges. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in stress granules, P bodies, and messenger RNA transport granules, we have developed and applied a variety of NMR methods for studies of condensed IDR states to provide insights into interactions driving and modulating phase separation. We identify ATP interactions with CAPRIN1 that can enhance or reduce phase separation. We also quantify specific side-chain and backbone interactions within condensed CAPRIN1 that define critical sequences for phase separation and that are reduced by O -GlcNAcylation known to occur during cell cycle and stress. This expanded NMR toolkit that has been developed for characterizing IDR condensates has generated detailed interaction information relevant for understanding CAPRIN1 biology and informing general models of phase separation, with significant potential future applications to illuminate dynamic structure-function relationships in other biological condensates., Competing Interests: The authors declare no competing interest.

Objective: This study aimed to compare measurements of right ventricular function using three-dimensional transesophageal echocardiography (3D TEE), and pulmonary artery catheters (PACs) in patients undergoing cardiac surgery. The authors examined the practicality of using the 3D TEE., Design: Prospective observational., Setting: Cardiac operating room at a single university hospital., Participants: All adult patients undergoing elective cardiac surgery at a single tertiary care university hospital over two years., Interventions: None., Measurements and Main Results: Right ventricular end-diastolic volume (RVEDV), right ventricular end-systolic volume (RVESV), stroke volume (SV), and right ventricular ejection fraction (RVEF) were measured with both 3D TEE and PACs. Assessments were performed using correlation coefficients, paired t tests, and Bland-Altman plots. Thirty-one patients participated in this study. Each measurement showed good agreement. RVEDV and RVESV were slightly lower on 3D TEE than on PAC (205.9 mL v 220.2 mL, p = 0.0018; 143.0 mL v 155.5 mL, p = 0.0143, respectively), whereas no significant differences were observed for SV and RVEF (31.0% v 31.1%, p = 0.0569; 61.6 mL v 66.9 mL, p = 0.92, respectively). Linear regression analysis showed high correlation between 3D TEE and PAC for RVEDV (r = 0.87) and RVESV (r = 0.81), and moderate correlation for SV (r = 0.67) and RVEF (r = 0.67). In the Bland-Altman plot, most patients were within the 95% limits of the agreement throughout all measurements., Conclusion: A high correlation was found between measurements made with a PAC and with 3D TEE in the assessment of right ventricular function. Three-dimensional TEE would be a potential alternative to PAC for assessment of right ventricular function during intraoperative periods., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins., Competing Interests: The authors declare no competing interest.

Background: Novel coronavirus disease 2019 (COVID-19) refers to infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen, and has spread to pandemic levels since its inception in December 2019. While several risk factors for severe presentation have been identified, the clinical course for end-stage renal disease (ESRD) patients on maintenance hemodialysis with COVID-19 has been unclear. Previous studies have revealed that some antiviral agents may be effective against COVID-19 in the general population, but the pharmacokinetics and pharmacodynamics of these agents in ESRD patients remain under investigation. Favipiravir, an antiviral agent developed for treatment of influenza, is one candidate treatment for COVID-19, but suitable dosages for patients with renal insufficiency are unknown. Here we provide a first report on the efficacy of favipiravir in a patient with ESRD undergoing hemodialysis., Case Presentation: The case involved a 52-year-old woman with COVID-19 who had been undergoing maintenance hemodialysis three times a week for 3 years due to diabetic nephropathy. She had initially been treated with lopinavir/ritonavir and ciclesonide for 5 days, but developed severe pneumonia requiring invasive positive-pressure ventilation. Those antiviral agents were subsequently switched to favipiravir. She recovered gradually, and after 2 weeks was extubated once the viral load of SARS-CoV-2 fell below the limit of detection. Although concentrations of several biliary enzymes were elevated, no major adverse events were observed., Conclusion: Favipiravir may be an effective option for the treatment of COVID-19-infected patients with ESRD.

The potassium ion (K + ) channel plays a fundamental role in controlling K + permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmembrane pore reportedly affect the gating transitions of K + channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K + channels, the structural mechanism by which these mutations cause K + channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectroscopy to investigate the structural mechanism underlying the decreased K + -permeation caused by disease-related mutations, using the prokaryotic K + channel KcsA. We demonstrated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K + -gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K + -gate is one of the fundamental mechanisms underlying the dysfunctions of K + channels caused by disease-related mutations.

Solution NMR spectroscopy is widely used to investigate the thermodynamics and kinetics of the binding of ligands to their biological receptors, as it provides detailed, atomistic information, potentially leading to microscopic affinities for each binding event, and, to the development of allosteric pathways describing how the binding at one site affects distal sites in the molecule. Importantly, weak interactions that are often invisible to other biophysical methods can also be probed. Methodological advancements in NMR have enabled the investigation of high molecular weight, homo-oligomeric complexes that bind multiple ligand molecules, with increasing numbers of studies of the structural dynamics and binding properties of these systems. It therefore becomes of interest to consider how binding and kinetics parameters can be extracted from experiments on these more complicated molecules. Here we present the theoretical framework for analyzing binding reactions of homo-oligomeric complexes by NMR, taking into account all of the chemical species in solution and their corresponding NMR observables. A number of simulations are presented to illustrate the utility of the derived expressions., Competing Interests: Declaration of Competing Interest The authors declare no competing conflicts of interest., (Copyright © 2020. Published by Elsevier Inc.)

Ras-related C3 botulinum toxin substrate 1 (Rac1) functions as a molecular switch by cycling between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state. An oncogenic mutant of Rac1, an N92I mutant, strongly promotes cell proliferation and subsequent oncogenic activities by facilitating the intrinsic GDP dissociation in the inactive GDP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the N92I mutant. We found that the static structure of the GDP binding site is not markedly perturbed by the mutation, but the overall conformational stability decreases in the N92I mutant, which then facilitates GDP dissociation by lowering the activation energy for the dissociation reaction. On the basis of these results, we proposed the activation mechanism of the N92I mutant, in which the decreased conformational stability plays important roles in its activation process.

Multidimensional nuclear magnetic resonance (NMR) spectroscopy has enabled detailed characterizations of protein structures and dynamics that are closely linked to functions. However, it leads to a large sensitivity loss in applications to high molecular weight proteins, which is caused by spin relaxation during the frequency discrimination period in the indirect dimension. Here, we describe a selective coherence transfer scheme, which enables us to selectively observe 1 H nuclei bonded to 15 N or 13 C nuclei with specified resonance frequencies. By utilizing this scheme, we achieved a 2.5- to 6-fold increase in signal height per unit of time with this scheme by avoiding the relaxation loss in the indirect dimension, as compared to the conventional two-dimensional heteronuclear correlation spectroscopy. We also demonstrated the effectiveness of this approach with applications to the membrane protein KirBac1.1, and characterized the functionally relevant conformational exchange process in both detergent micelles and a reconstituted membrane environment, corresponding to the apparent molecular masses of 220 kDa and 300 kDa, respectively., (Copyright © 2019 Elsevier Inc. All rights reserved.)

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.

Herein, we report the case of a 49-year-old man with a potentially fatal allergy to propofol and remifentanil who underwent awake minimally invasive mitral valve surgery with cardiopulmonary bypass using thoracic epidural anesthesia, without the need for endotracheal general anesthesia. The aim was the management of spontaneous respiration during cardiopulmonary bypass surgery in an awake patient., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Ras-related C3 botulinum toxin substrate 1 (Rac1) plays critical roles in the maintenance of cell morphology by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Rac1 P29S mutant is known to strongly promote oncogenesis by facilitating its intrinsic GDP dissociation and thereby increasing the level of the GTP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the oncogenic P29S mutant. We demonstrate that the conformational landscape is markedly altered in the mutant, and the preexisting equilibrium is shifted toward the conformation with reduced affinity for Mg 2+ , a cofactor that is critical for maintaining stable GDP binding. Our results suggest that the alternation of the preexisting conformational equilibrium of proteins is one of the fundamental mechanisms underlying their oncogenic activities.

Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system., Competing Interests: The authors declare no conflict of interest.

Objective: To evaluate the morphologic changes of the mitral annulus using 3-dimensional transesophageal echocardiography during heart displacement to expose the anastomosis site in off-pump coronary artery bypass surgery (OPCAB)., Design: Prospective case series., Setting: Single center, university hospital., Participants: The study comprised 34 consecutive patients who underwent OPCAB of the left circumflex artery (LCX) and the right coronary artery (RCA)., Interventions: None., Measurements and Main Results: Mitral annulus parameters were measured using the Mitral Valve Quantification program after sternotomy (physiologic position) and during displacement of the heart to expose the LCX (LCX position) and the RCA (RCA position). The height of the mitral annulus was significantly lower in the LCX (5.76 ± 0.90 mm) and RCA (5.92 ± 0.97 mm) positions than in the physiologic position (6.96 ± 0.99 mm; both p < 0.0001). The percent change in the height of the mitral annulus was significantly greater in the mitral regurgitation group than in the mitral regurgitation nondeterioration group when in the LCX (-16.3% ± 6.0% v -11.9% ± 3.3%, p = 0.0203) and RCA (-16.9% ± 6.3% v -12.1% ± 3.8%, p = 0.0207) positions. The anteroposterior and intercommissural diameters, annulus perimeter, and surface area of the mitral annulus did not differ significantly among all heart positions., Conclusions: The mitral annulus flattened and lost its saddle shape without expanding while in the LCX and RCA positions. The greater percent change in the height of the mitral annulus may aggravate mitral regurgitation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The gating of potassium ion (K + ) channels is regulated by various kinds of protein-protein interactions (PPIs). Structural investigations of these PPIs provide useful information not only for understanding the gating mechanisms of K + channels, but also for developing the pharmaceutical compounds targeting K + channels. Here, we describe a nuclear magnetic resonance spectroscopic method, termed the cross saturation (CS) method, to accurately determine the binding surfaces of protein complexes, and its application to the investigation of the interaction between a G protein-coupled inwardly rectifying K + channel and a G protein α subunit.

End-stage renal disease (ESRD) is associated with significant alterations in cardiovascular function; homeostasis of body fluid, electrolytes, and acid-base equilibrium; bone metabolism, erythropoiesis; and blood coagulation. The prevalence of ESRD is increasing rapidly worldwide, as is the number of patients requiring surgery under general anesthesia. Patients with ESRD have significantly higher risks of perioperative morbidity and mortality due to multiple comorbidities. The perioperative management of patients with ESRD under general anesthesia therefore requires special considerations and a careful multidisciplinary approach. In this review, the authors summarize the available literature to address common issues related to patients with ESRD and discuss the best perioperative approach for this patient subgroup., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Laser speckle flowgraphy (LSFG) is an ophthalmologic equipment that qualitatively detects the blood flow of the optic nerve head, which is known to be related with cerebral microcirculation. LSFG can also measure the mean blur rate, which quantitatively calculates the blood flow. We aimed to assess the utility of LSFG in the evaluation of cerebral perfusion during aortic surgery under hypothermic circulatory arrest with retrograde and antegrade cerebral perfusion. Two patients underwent total arch replacement for aneurysm. The blood flow of the optic nerve head was monitored with LSFG and the mean blur rate value was measured during the surgery. The LSFG could detect the blood flow quantitatively in the optic nerve head during both retrograde and antegrade cerebral perfusion; and the value was correlated with rSO2 value.

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Chemical exchange processes of proteins on the order of microseconds (μs) to milliseconds (ms) play critical roles in biological functions. Developments in methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), which observes the slowly relaxing multiple quantum (MQ) coherences, have enabled the studies of biologically important large proteins. However, the analyses of μs to ms chemical exchange processes based on the methyl-TROSY principle are still challenging, because the interpretation of the chemical exchange contributions to the MQ relaxation profiles is complicated, as significant chemical shift differences occur in both (1)H and (13)C nuclei. Here, we report a new methyl-based NMR method for characterizing chemical exchanges, utilizing differential MQ relaxation rates and a heteronuclear double resonance pulse technique. The method enables quantitative evaluations of the chemical exchange processes, in which significant chemical shift differences exist in both the (1)H and (13)C nuclei. The versatility of the method is demonstrated with the application to KirBac1.1, with an apparent molecular mass of 200 kDa.

Purpose: The aim of this study was to investigate fluid loading-induced changes in left ventricular end-diastolic volume (LVEDV) and stroke volume variability (SVV) in patients with end-stage renal disease (ESRD) using real-time three-dimensional transesophageal echocardiography and the Vigileo-FloTrac system., Patients and Methods: After obtaining ethics committee approval and informed consent, 28 patients undergoing peripheral vascular procedures were studied. Fourteen patients with ESRD on hemodialysis (HD) were assigned to the HD group and 14 patients without ESRD were assigned to the control group. Institutional standardized general anesthesia was provided in both groups. SVV was measured using the Vigileo-FloTrac system. Simultaneously, a full-volume three-dimensional transesophageal echocardiography dataset was acquired to measure LVEDV, left ventricular end-systolic volume, and left ventricular ejection fraction. Measurements were obtained before and after loading 500 mL hydroxyethyl starch over 30 minutes in both groups., Results: In the control group, intravenous colloid infusion was associated with a significant decrease in SVV (13.8%±2.6% to 6.5%±2.6%, P<0.001) and a significant increase in LVEDV (83.6±23.4 mL to 96.1±28.8 mL, P<0.001). While SVV significantly decreased after infusion in the HD group (16.2%±6.0% to 6.2%±2.8%, P<0.001), there was no significant change in LVEDV., Conclusion: Our preliminary data suggest that fluid responsiveness can be assessed not by LVEDV but also by SVV due to underlying cardiovascular pathophysiology in patients with ESRD.

Purpose: The aim of this clinical trial was to investigate changes in stroke volume variability (SVV) and left ventricular end-diastolic volume (LVEDV) after a fluid bolus of crystalloid or colloid using real-time three-dimensional transesophageal echocardiography (3D-TEE) and the Vigileo-FloTrac™ system., Materials and Methods: After obtaining Institutional Review Board approval, and informed consent from the research participants, 22 patients undergoing scheduled peripheral vascular bypass surgery were enrolled in the study. The patients were randomly assigned to receive 500 mL of hydroxyethyl starch (HES; HES group, n=11) or normal saline (Saline group, n=11) for fluid replacement therapy. SVV was measured using the Vigileo-FloTrac system. LVEDV, stroke volume, and cardiac output were measured by 3D-TEE. The measurements were performed over 30 minutes before and after the fluid bolus in both groups., Results: SVV significantly decreased after fluid bolus in both groups (HES group, 14.7%±2.6% to 6.9%±2.7%, P<0.001; Saline group, 14.3%±3.9% to 8.8%±3.1%, P<0.001). LVEDV significantly increased after fluid loading in the HES group (87.1±24.0 mL to 99.9±27.2 mL, P<0.001), whereas no significant change was detected in the Saline group (88.8±17.3 mL to 91.4±17.6 mL, P>0.05). Stroke volume significantly increased after infusion in the HES group (50.6±12.5 mL to 61.6±19.1 mL, P<0.01) but not in the Saline group (51.6±13.4 mL to 54.1±12.8 mL, P>0.05). Cardiac output measured by 3D-TEE significantly increased in the HES group (3.5±1.1 L/min to 3.9±1.3 L/min, P<0.05), whereas no significant change was seen in the Saline group (3.4±1.1 L/min to 3.3±1.0 L/min, P>0.05)., Conclusion: Administration of colloid and crystalloid induced similar responses in SVV. A higher plasma-expanding effect of HES compared to normal saline was demonstrated by the significant increase in LVEDV.

Background: Most of the thermometers used during operations are invasive and non-hygienic. The usefulness of a noncontact continuous tympanic thermometer under general anesthesia has been reported. We evaluated the usefulness of a noncontact continuous tympanic thermometer in patients undergoing cardiac surgery using an artificial heart-lung machine., Methods: Twenty patients scheduled to undergo cardiac surgery using an artificial heart-lung machine were selected for this study. After induction of general anesthesia, thermistor probes were inserted into the rectum and esophagus for measurements of rectal and esophageal temperatures, respectively. A noncontact continuous tympanic thermometer was inserted into the ear canal on the right side. These temperatures were monitored and recorded at one-minute intervals. Regression analysis and Bland-Altman analysis were used to compare the data (tympanic/rectal temperatures) with esophageal temperature as a core temperature., Results: Tympanic temperature showed a good correlation with esophageal temperature (r=0.983, P<0.05). Rectal temperature also showed a good correlation with esophageal temperature (r=0.923, P<0.05), but the coefficient of correlation was low compared to that of tympanic temperature. The mean difference between tympanic temperature and esophageal temperature was -0.022 degrees C, and standard deviation (SD) was 0.395 degrees C. The mean difference between rectal temperature and esophageal temperature was -0.299 degrees C, and standard deviation (SD) was 0.838 degrees C., Conclusions: A noncontact continuous tympanic thermometer is useful for measurement of core temperature during cardiac surgery using an artificial heart-lung machine.

Study Objective: To evaluate the analgesia following cesarean delivery and the frequency of side effects of intrathecal morphine when combined with a continuous epidural infusion., Design: Randomized, double-blinded study., Setting: University hospital., Patients: 76 ASA physical status I and II term parturients undergoing cesarean delivery with combined spinal-epidural anesthesia., Interventions: Patients were randomized to one of three groups to receive 0, 50, or 100 microg (Group 0, Group 50, and Group 100, respectively) intrathecal morphine in addition to 8 mg of hyperbaric bupivacaine. Each patient received a continuous epidural infusion of 0.2% ropivacaine at the rate of 6 mL/hr., Measurements: 24-hour visual analog pain scores (VAPS), number of patients who requested rescue analgesics, frequency of requests for rescue analgesics per patient, and time interval before the first request for rescue analgesics were recorded. Frequency of pruritus and postoperative nausea and vomiting (PONV) were also recorded., Main Results: Group 50 and Group 100 patients exhibited lower VAPS and longer time intervals before the first request for rescue analgesics, and they requested rescue analgesics less frequently than Group 0 patients. The frequency of pruritus was significantly higher in Group 100 than Group 0. The groups did not differ with regard to PONV., Conclusions: 50 microg and 100 microg of intrathecal morphine improve analgesia when combined with a continuous epidural infusion of 0.2% ropivacaine (6 mL/hr) after cesarean delivery. 50 microg of intrathecal morphine is associated with a low frequency of side effects such as pruritus and PONV., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

An 11-year-old boy with X-linked alpha-thalassaemia/ mental retardation symptom underwent gastrostomy. After the surgical procedure, insertion of a central venous (CV) catheter via the left subclavian vein was performed under X-ray radioscopy. However, the catheter did not move toward the right side of the heart shadow but descended caudally through the left side of the heart shadow. Transesophageal echocardiography (TEE) showed thickening of the coronary sinus, suggesting the existence of a persistent left superior vena cava (PLSVC). Contrast echocardiography using infusion of agitated saline into the CV catheter was performed. Microbubbles flowing from the coronary sinus into the right atrium were observed, and a diagnosis of PLSVC was made. TEE is useful for diagnosis of PLSVC when passage of a CV catheter via the left subclavian vein or left internal jugular vein is incorrect in a pediatric patient.

Background: The PENTAX-AWS (AWS), combined with PBLADE is a novel tracheal intubation device which allows indirect visualization of the vocal cords without the alignment of the oral, pharyngeal axes required for direct laryngoscopy. Intubation procedure can be monitored on a built-in CCD monitor., Methods: We compared the laryngeal view obtained with the Macintosh laryngoscope with that obtained with the AWS in 100 patients scheduled for elective anesthesia. Laryngeal view was determined by Cormack & Lehane classification modified by Cook, first using the Macintosh laryngoscope with the patient's head and neck in the sniffing position, followed by the AWS with the head and neck in a neutral position. The time taken to pass the endotracheal tube, the incidence of tube impingement on the arytenoids and the total number of intubation attempts were also recorded., Results: The best laryngeal view obtained with the Macintosh laryngoscope in 100 cases were Grade I: 65, IIa: 16, IIb: 8, IIIa: 6, IIIb: 5, IV: 0. With AWS, the laryngeal views obtained were all grade I. The total time to pass the tube with the AWS was 19.5 +/- 7.9 s and the number of intubation attempts was 1.05 +/- 0.2. The Cormack grade obtained with the Macintosh laryngoscope did not affect the AWS view. The tube touched the right arytenoid in 5 cases but successful intubation was achieved by adjusting the blade direction., Conclusions: We conclude that the PENTAX-AWS provides a better laryngeal view than the Macintosh laryngoscope and facilitates easier intubation under vision in a higher proportion of patients.