Ensitrelvir in patients with SARS-CoV-2: A retrospective chart review.

Antivirals with proven effectiveness against the Omicron SARS-CoV-2 variant are required for COVID-19 treatment in hospitalized patients, particularly those with severe underlying conditions. Ensitrelvir, a 3C-like protease inhibitor, received emergency approval in Japan in November 2022, based on evidence of rapid symptom resolution in non-hospitalized patients, but confirmation of its effectiveness in hospitalized patients is lacking. This retrospective chart review reports outcomes for all patients who received ensitrelvir whilst hospitalized with SARS-CoV-2 infection at Rinku General Medical Center, Japan (November 2022-April 2023). Thirty-two hospitalized patients received 5 days of ensitrelvir treatment (375 mg loading dose, 125 mg as maintenance dose). Patients' mean age was 73.5 years and most had mild COVID-19. Patients exhibited various underlying diseases, most commonly hypertension (78.1%) and chronic kidney disease (25.0%). Seven (21.9%) patients were on hemodialysis. The most common concomitant medications were antihypertensives (59.4%) and corticosteroids (31.3%); 2 (6.3%) patients were being treated with rituximab; 28 (87.5%) patients had viral persistence following pre-treatment by remdesivir. Following ensitrelvir treatment, viral clearance was recorded in 18 (56.3%) patients by Day 6 and 25 (78.1%) patients at final measurement. All patients experienced clinical improvement as assessed by the investigator at Day 5. No intensive care unit admissions or deaths due to COVID-19 occurred. No new safety signals were observed. In conclusion, positive virological outcomes were observed following ensitrelvir treatment, in hospitalized patients with SARS-CoV-2 in a real-world setting, including high-risk patients, who failed previous antiviral therapy. These results require confirmation in more extensive studies. TRIAL REGISTRATION: UMIN000051300.

Baloxavir Marboxil for Prophylaxis against Influenza in Household Contacts.

BACKGROUND: Baloxavir marboxil (baloxavir) is a polymerase acidic protein (PA) endonuclease inhibitor with clinical efficacy in the treatment of uncomplicated influenza, including in outpatients at increased risk for complications. The postexposure prophylactic efficacy of baloxavir in the household setting is unclear. METHODS: We conducted a multicenter, double-blind, randomized, placebo-controlled trial to evaluate the postexposure prophylactic efficacy of baloxavir in household contacts of index patients with confirmed influenza during the 2018-2019 season in Japan. The participants were assigned in a 1:1 ratio to receive either a single dose of baloxavir or placebo. The primary end point was clinical influenza, as confirmed by reverse-transcriptase-polymerase-chain-reaction testing, over a period of 10 days. The occurrence of baloxavir-selected PA substitutions associated with reduced susceptibility was assessed. RESULTS: A total of 752 household contacts of 545 index patients were randomly assigned to receive baloxavir or placebo. Among the index patients, 95.6% had influenza A virus infection, 73.6% were younger than 12 years of age, and 52.7% received baloxavir. Among the participants who could be evaluated (374 in the baloxavir group and 375 in the placebo group), the percentage in whom clinical influenza developed was significantly lower in the baloxavir group than in the placebo group (1.9% vs. 13.6%) (adjusted risk ratio, 0.14; 95% confidence interval [CI], 0.06 to 0.30; P<0.001). Baloxavir was effective in high-risk, pediatric, and unvaccinated subgroups of participants. The risk of influenza infection, regardless of symptoms, was lower with baloxavir than with placebo (adjusted risk ratio, 0.43; 95% CI, 0.32 to 0.58). The incidence of adverse events was similar in the two groups (22.2% in the baloxavir group and 20.5% in the placebo group). In the baloxavir group, the viral PA substitutions I38T/M or E23K were detected in 10 (2.7%) and 5 (1.3%) participants, respectively. No transmission of these variants from baloxavir-treated index patients to participants in the placebo group was detected; however, several instances of transmission to participants in the baloxavir group could not be ruled out. CONCLUSIONS: Single-dose baloxavir showed significant postexposure prophylactic efficacy in preventing influenza in household contacts of patients with influenza. (Funded by Shionogi; Japan Primary Registries Network number, JapicCTI-184180.).

Validation of actigraphy in hospitalised newborn infants using video polysomnography.

Actigraphy has been established as a reliable sleep assessment tool in adults; however, its utility in newborns remains unknown. Validation of actigraphy in newborns may provide a significant insight into the physiological and pathological acquisition process of mature diurnal sleep patterns and subsequent morbidities in both newborns and their mothers. Thus, the present study aimed to evaluate the accuracy of sleep-wake detection by overnight actigraphy in a cohort of newborns. Simultaneous recording of polysomnography and actigraphy data was performed in 40 newborns admitted to a tertiary neonatal intensive care unit (NICU). A mixed-effects logistic regression model to explain the sleep state identified by polysomnography was employed using the actigraphic activity score as a fixed independent variable and the individual newborn's identity as a random effect. To evaluate the usefulness of the actigraphic activity score as a surrogate marker of sleep, a receiver operating characteristic (ROC) curve analysis was performed using the variables that were used in the mixed-effects logistic regression model, and the area under the curve (AUC) was assessed. The results showed that polysomnography-determined sleep epochs were associated with a smaller activity index on actigraphy (odds ratio per 10 activity indices increase 0.81, 95% confidence interval [CI] 0.79-0.84). The AUC for the ROC curve was 0.87 (95% CI 0.87-0.88, range 0.54-0.99). An activity score of 124 showed the maximum overall accuracy (90.2%, 95% CI 87.7-92.1). Our present study suggests that sleep-wake states of NICU-hospitalised newborns can be precisely determined using actigraphy on the ankle.

Serum matrix metalloproteinase-7 in biliary atresia: A Japanese multicenter study.

BACKGROUND: Biliary atresia (BA) is among the commonest indications for liver transplantation (LT) in children. We examined whether serum matrix metalloproteinase-7 (MMP-7) is useful for diagnosis of BA in Japanese infants, and whether serum MMP-7 concentrations before and after Kasai portoenterostomy (KP) predicted LT within a year. METHODS: Subjects under 6 months old at eight pediatric centers in Japan were enrolled retrospectively, including patients with cholestasis and normal controls (NC) without liver disease. Patients with cholestasis were divided into groups representing BA versus cholestasis from other causes (non-BA). Serum samples were collected from patients with BA at diagnosis and 1 and 4 weeks after KP, as well as from non-BA and NC. RESULTS: Serum MMP-7 concentrations were significantly higher in BA at diagnosis (median, 89.1 ng/ml) than in non-BA (11.0; p < 0.001) or NC (10.3; p < 0.001). Receiver operating characteristic (ROC) analysis of MMP-7 for BA versus non-BA yielded an area under the ROC curve of 0.99 (95% confidence interval, 0.96-1.00). An optimal cut-off value of 18.6 ng/ml for serum MMP-7 in diagnosing BA demonstrated sensitivity and specificity of 100% and 90%, respectively. Serum MMP-7 before and 1 week and 4 weeks after KP did not differ significantly between BA requiring only KP and BA requiring LT after KP. CONCLUSION: Serum MMP-7 is a useful marker for diagnosis of BA in Japanese infants, but it could not predict LT within a year.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Clinical and virological outcomes with baloxavir compared with oseltamivir in pediatric patients aged 6 to < 12 years with influenza: an open-label, randomized, active-controlled trial protocol.

BACKGROUND: Children with influenza virus infections are prone to complications and are common sources of influenza transmission. Baloxavir marboxil inhibits cap-dependent endonuclease and was approved for influenza treatment in adolescent, adult, and pediatric patients in Japan. The miniSTONE-2 study included pediatric patients with influenza (1 to < 12 years) and demonstrated similar median times to alleviation of signs and symptoms of influenza with a single dose of baloxavir granules (weight < 20 kg: 2 mg/kg, ≥ 20 kg: 40 mg) and oseltamivir. Although the baloxavir dose in miniSTONE-2 was higher than the Japanese-approved dose, baloxavir exposure in miniSTONE-2 was similar to Japanese pediatric patients who receive the Japanese-approved dose. This study will be the first randomized active-controlled study in pediatric patients with influenza using the Japanese-approved dose of baloxavir. METHODS: This is a multicenter, open-label, randomized, active-controlled trial in which 200 Japanese subjects aged 6 to < 12 years with influenza virus infection are randomly allocated (2:1) to a single dose of baloxavir at the approved dose in Japan (weight ≥ 10 to < 20 kg: 10 mg, ≥ 20 to < 40 kg: 20 mg, ≥ 40 kg: 40 mg) or oseltamivir twice daily for 5 days. The primary clinical endpoint is the time to illness alleviation of influenza, from administration of baloxavir or oseltamivir until the following criteria were met and sustained for at least 21.5 h (24 h-10%): cough and nasal discharge/nasal congestion rated as absent or mild axillary body temperature < 37.5 °C. The primary analysis population is the intention-to-treat infected population, which includes all pediatric subjects who receive at least one dose of study drug and have confirmed influenza virus infection by reverse transcription-polymerase chain reaction. The safety population includes all subjects who receive at least one dose of study drug. DISCUSSION: No comparative studies have been conducted to confirm the efficacy and safety of baloxavir versus a comparator in pediatric patients with influenza infection in Japan. The outcomes from this trial will provide evidence on the efficacy and safety of baloxavir as an antiviral treatment option for Japanese pediatric patients with influenza infection. Trial registration Japan Registry of Clinical Trials: jRCTs011200011. Registered November 2020. ( https://rctportal.niph.go.jp/en/ ).

Revisions of clinical protocols using the Plan Do Check Act cycle improved outcomes of extremely preterm infants at 2 years.

AIM: Clinical quality improvement is often cumbersome due to established protocols. We aimed to investigate whether outcomes of preterm infants improve with protocol revisions using iteration cycles. METHODS: Preterm infants born <28 weeks gestation between January 2006 and December 2015 were retrospectively analysed. Protocols were revised using Plan Do Check Act cycle. Death and serious adverse events at term were reviewed in six-monthly quality improvement meetings. Adverse outcome of death or motor/sensory impairments at two years was compared before and after two major protocol changes, which were implemented in January 2008 and January 2012. RESULTS: Based on the appraisal for period 2006-2007, strategies for surfactant, narcotics, parenteral nutrition, respiratory gas humidity and prophylactic indomethacin and antibiotics were changed for period 2008-2011. For period 2012-2015, stabilisation of infants was accelerated via very early catheterisation. Of 162 infants (84 males, 25.5 ± 1.5 weeks gestation) within the whole cohort, 63 developed adverse outcomes, which were fewer for periods 2008-2011 (p = 0.013) and 2012-2015 (p = 0.035) compared with period 2006-2007 (adjusted for gestational age, Apgar scores and sex). CONCLUSION: Careful bottom-up revisions of protocols using iteration cycles, accounting for local settings, successfully improved the outcomes of preterm infants.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor.

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained.

We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation Tm is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose Tm reaches ∼100 °C is also discovered.

Hydration properties of a protein at low and high pressures: Physics of pressure denaturation.

Using experimentally determined structures of ubiquitin at 1 and 3000 bar, we generate sufficiently large ensembles of model structures in the native and pressure-induced (denatured) states by means of molecular dynamics simulations with explicit water. We calculate the values of a free-energy function (FEF), which comprises the hydration free energy (HFE) and the intramolecular (conformational) energy and entropy, for the two states at 1 and 3000 bar. The HFE and the conformational entropy, respectively, are calculated using our statistical-mechanical method, which has recently been shown to be accurate, and the Boltzmann-quasi-harmonic method. The HFE is decomposed into a variety of physically insightful components. We show that the FEF of the native state is lower than that of the denatured state at 1 bar, whereas the opposite is true at 3000 bar, thus being successful in reproducing the pressure denaturation. We argue that the following two quantities of hydration play essential roles in the denaturation: the WASA-dependent term in the water-entropy loss upon cavity creation for accommodating the protein (WASA is the water-accessible surface area of the cavity) and the protein-water Lennard-Jones interaction energy. At a high pressure, the mitigation of the serious water crowding in the system is the most important, and the WASA needs to be sufficiently enlarged with the increase in the excluded-volume being kept as small as possible. The denatured structure thus induced is characterized by the water penetration into the protein interior. The pressure denaturation is accompanied by a significantly large gain of water entropy.

How Does the Recently Discovered Peptide MIP Exhibit Much Higher Binding Affinity than an Anticancer Protein p53 for an Oncoprotein MDM2?

An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.

Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability.

Although the two membrane proteins, thermophilic rhodopsin (TR) and xanthorhodopsin (XR), share a high similarity in amino-acid sequence and an almost indistinguishable three-dimensional structure, TR is much more thermostable than XR. This is counterintuitive also because TR possesses only a smaller number of intramolecular hydrogen bonds (HBs) than XR. Here we investigate physical origins of the remarkable difference between XR and TR in the stability. Our free-energy function (FEF) is improved so that not only the portion within the transmembrane (TM) region but also the extracellular and intracellular portions within the water-immersed (WI) regions can be considered in assessing the stability. The assessment is performed on the basis of the FEF change upon protein folding, which is calculated for the crystal structure of XR or TR. Since the energetics within the TM region is substantially different from that within the WI regions, we determine the TM and WI portions of XR or TR by analyzing the distribution of water molecules using all-atom molecular dynamics simulations. The energetic component of the FEF change consists of a decrease in energy arising from the formation of intramolecular HBs and an increase in energy caused by the break of protein-water HBs referred to as "energetic dehydration penalty." The entropic component is a gain of the translational, configurational entropies of hydrocarbon groups within the lipid bilayer and of water molecules. The entropic component is calculated using the integral equation theory combined with our morphometric approach. The energetic one is estimated by a simple but physically reasonable method. We show that TR is much more stable than XR for the following reasons: The decrease in energy within the TM region is larger, and the energetic dehydration penalty within the WI regions is smaller, leading to higher energetic stabilization, and tighter packing of side chains accompanying the association of seven helices confers higher entropic stabilization on TR.

Reduced density profile of small particles near a large particle: Results of an integral equation theory with an accurate bridge function and a Monte Carlo simulation.

Solute-solvent reduced density profiles of hard-sphere fluids were calculated by using several integral equation theories for liquids. The traditional closures, Percus-Yevick (PY) and the hypernetted-chain (HNC) closures, as well as the theories with bridge functions, Verlet, Duh-Henderson, and Kinoshita (named MHNC), were used for the calculation. In this paper, a one-solute hard-sphere was immersed in a one-component hard-sphere solvent and various size ratios were examined. The profiles between the solute and solvent particles were compared with those calculated by Monte Carlo simulations. The profiles given by the integral equations with the bridge functions were much more accurate than those calculated by conventional integral equation theories, such as the Ornstein-Zernike (OZ) equation with the PY closure. The accuracy of the MHNC-OZ theory was maintained even when the particle size ratio of solute to solvent was 50. For example, the contact values were 5.7 (Monte Carlo), 5.6 (MHNC), 7.8 (HNC), and 4.5 (PY), and the first minimum values were 0.48 (Monte Carlo), 0.46 (MHNC), 0.54 (HNC), and 0.40 (PY) when the packing fraction of the hard-sphere solvent was 0.38 and the size ratio was 50. The asymptotic decay and the oscillation period for MHNC-OZ were also very accurate, although those given by the HNC-OZ theory were somewhat faster than those obtained by Monte Carlo simulations.

An accurate and rapid method for calculating hydration free energies of a variety of solutes including proteins.

A new method is developed for calculating hydration free energies (HFEs) of polyatomic solutes. The solute insertion is decomposed into the creation of a cavity in water matching the geometric characteristics of the solute at the atomic level (process 1) and the incorporation of solute-water van der Waals and electrostatic interactions (process 2). The angle-dependent integral equation theory combined with our morphometric approach and the three-dimensional interaction site model theory are applied to processes 1 and 2, respectively. Neither a stage of training nor parameterization is necessitated. For solutes with various sizes including proteins, the HFEs calculated by the new method are compared to those obtained using a molecular dynamics simulation based on solution theory in energy representation (the ER method developed by Matubayasi and co-workers), currently the most reliable tool. The agreement is very good especially for proteins. The new method is characterized by the following: The calculation can rapidly be finished; a solute possessing a significantly large total charge can be handled without difficulty; and since it yields not only the HFE but also its many physically insightful energetic and entropic components, it is best suited to the elucidation of mechanisms of diverse phenomena such as the receptor-ligand binding, different types of molecular recognition, and protein folding, denaturation, and association.

Mechanism of protein-RNA recognition: analysis based on the statistical mechanics of hydration.

We investigate the RBD1-r(GUAGU) binding as a case study using all-atom models for the biomolecules, molecular models for water, and the currently most reliable statistical-mechanical method. RBD1 is one of the RNA-binding domains of mammalian Musashi1 (Msi1), and r(GUAGU) contains the minimum recognition sequence for Msi1, r(GUAG). We show that the binding is driven by a large gain of configurational entropy of water in the entire system. It is larger than the sum of conformational-entropy losses for RBD1 and r(GUAGU). The decrease in RBD1-r(GUAGU) interaction energy upon binding is largely cancelled out by the increase in the sum of RBD1-water, r(GUAGU)-water, and water-water interaction energies. We refer to this increase as "energetic dehydration". The decrease is larger than the increase for the van der Waals component, whereas the opposite is true for the electrostatic component. We give a novel reason for the empirically known fact that protein residues possessing side chains with positive charges and with flat moieties frequently appear within protein-RNA binding interfaces. A physical picture of the general protein-RNA binding mechanism is then presented. To achieve a sufficiently large water-entropy gain, shape complementarity at the atomic level needs to be constructed by utilizing the stacking and sandwiching of flat moieties (aromatic rings of the protein and nucleobases of RNA) as fundamental motifs. To compensate for electrostatic energetic dehydration, charge complementarity becomes crucial within the binding interface. We argue the reason why the RNA recognition motif (RRM) is the most ubiquitous RNA binding domain.

Statistical thermodynamics for the unexpectedly large difference between disaccharide stereoisomers in terms of solubility in water.

We unravel the physical origins of the large difference between cellobiose and maltose, which consist of two ß-1,4 and α-1,4 linked d-glucose units, respectively, in terms of the solubility in water. We construct a thermodynamic theory where the chemical-potential difference between disaccharides in water and in vacuum is identified as the key free-energy function. Its energetic and entropic components are calculated for cellobiose and maltose by statistical-mechanical theories for solute hydration. The disaccharide structures are taken into account at the atomic level and a molecular model is adopted for water. Molecular dynamics simulations are used to account for the conformational fluctuation of a disaccharide molecule, which also enables us to estimate the conformational entropy. We show that the cellobiose/maltose solubility ratio calculated is in good agreement with the experimental value. The solubility becomes much lower for cellobiose due to conformational-entropy and water-entropy effects. The former effect is relevant to higher stability of the intramolecular hydrogen bond between oxygen atoms in the six-membered ring and in the neighboring hydroxyl group: the hydration alters the fluctuation of a molecular conformation to a larger or less regular one, but the degree of this alteration is smaller. The latter effect is attributed to more separation of two hydroxymethyl groups in a molecule, causing lower probability of the overlap of excluded volumes generated by the groups for water molecules. We suggest that physicochemical properties of disaccharides in water become variable depending on the stereoisomerism through hydration effects and the origins of the variety are entropic.

Universal effects of solvent species on the stabilized structure of a protein.

We investigate the effects of solvent specificities on the stability of the native structure (NS) of a protein on the basis of our free-energy function (FEF). We use CPB-bromodomain (CBP-BD) and apoplastocyanin (apoPC) as representatives of the protein universe and water, methanol, ethanol, and cyclohexane as solvents. The NSs of CBP-BD and apoPC consist of 66% α-helices and of 35% ß-sheets and 4% α-helices, respectively. In order to assess the structural stability of a given protein immersed in each solvent, we contrast the FEF of its NS against that of a number of artificially created, misfolded decoys possessing the same amino-acid sequence but significantly different topology and α-helix and ß-sheet contents. In the FEF, we compute the solvation entropy using the morphometric approach combined with the integral equation theories, and the change in electrostatic (ES) energy upon the folding is obtained by an explicit atomistic but simplified calculation. The ES energy change is represented by the break of protein-solvent hydrogen bonds (HBs), formation of protein intramolecular HBs, and recovery of solvent-solvent HBs. Protein-solvent and solvent-solvent HBs are absent in cyclohexane. We are thus able to separately evaluate the contributions to the structural stability from the entropic and energetic components. We find that for both CBP-BD and apoPC, the energetic component dominates in methanol, ethanol, and cyclohexane, with the most stable structures in these solvents sharing the same characteristics described as an association of α-helices. In particular, those in the two alcohols are identical. In water, the entropic component is as strong as or even stronger than the energetic one, with a large gain of translational, configurational entropy of water becoming crucially important so that the relative contents of α-helix and ß-sheet and the content of total secondary structures are carefully selected to achieve sufficiently close packing of side chains. If the energetic component is excluded for a protein in water, the priority is given to closest side-chain packing, giving rise to the formation of a structure with very low α-helix and ß-sheet contents. Our analysis, which requires minimal computational effort, can be applied to any protein immersed in any solvent and provides robust predictions that are quite consistent with the experimental observations for proteins in different solvent environments, thus paving the way toward a more detailed understanding of the folding process.

Identification of thermostabilizing mutations for a membrane protein whose three-dimensional structure is unknown.

We recently developed a physics-based method for identifying thermostabilizing mutations of a membrane protein. The method uses a free-energy function F where the importance of translational entropy of hydrocarbon groups within the lipid bilayer is emphasized. All of the possible mutations can rapidly be examined. The method was illustrated for the adenosine A2a receptor (A2a R) whose three-dimensional (3D) structure experimentally determined was utilized as the wild-type structure. Nine single mutations and a double mutation predicted to be stabilizing or destabilizing were checked by referring to the experimental results: The success rate was remarkably high. In this work, we postulate that the 3D structure of A2a R is unknown. We construct candidate models for the 3D structure using the homology modeling and select the model giving the lowest value to the change in F on protein folding. The performance achieved is only slightly lower than that in the recent work. © 2016 Wiley Periodicals, Inc.

Unified elucidation of the entropy-driven and -opposed hydrophobic effects.

The association of nonpolar solutes is generally believed to be entropy driven, which has been shown to be true for the contact of small molecules, ellipsoids, and plates. However, it has been reported with surprise that a model cavity-ligand binding is entropy opposed. How can these apparently conflicting behaviors be elucidated? Here, we calculate the potential of mean force between hard-sphere solutes with various diameters in water and its entropic and enthalpic components using a statistical-mechanical theory for molecular liquids. It is shown that there is a very wide region where both of the two components are negative and large with the entropy-enthalpy compensation. Even for spheres, their contact is weakly entropy opposed when they are medium-sized. The entropic component (EC) is decomposed into physically insightful constituents with the aid of our morphometric approach. They provide us with useful information on the signs and magnitudes of contributions from the structural difference between the water near a single solute surface and that within the space confined between two solute surfaces and from the total volume available for the translational displacement of water molecules in the system. The decomposition enables us to identify the essential factors for discussing the EC: the hydrogen-bonding properties and density structure of the water within the confined space and the degree of water crowding in the bulk. These are largely dependent on the geometric characteristics of the solute pair such as solute shape, size, and intersolute distance. Both of the entropy-driven and -opposed hydrophobic effects can be explained within the same theoretical framework.

Dynamic manipulation of the local pH within a nanopore triggered by surface-induced phase transition.

Manipulating the local pH within nanoconfinement is essential in nanofluidics technology and its applications. Since the conventional strategy utilizes the overlapping of an electric double layer formed for charge compensation by protons near a negatively charged pore-wall surface, pH variation within a pore is limited to the acidic side. To achieve the variation at the alkaline side, we developed a system comprising a hydrophobic pore-wall surface and aqueous solution containing hydrophobic cations. Beyond a threshold cation concentration, a nanopore is filled with the second phase where the cations are remarkably enriched due to surface-induced phase transition (SIFT) originating from the hydrophobic effect. It is accompanied by the enrichment of coexisting anions. We experimentally show that pH in the second phase is much higher than in the bulk solution. Electrochemical measurements strongly suggest that the pH value can be increased from 4.8 to 10.7 within a 10 nm nanopore in the most significant case. This is ascribed to the enrichment of hydroxide anions. We argue that the extent and rate of pH variation are controlled as desired.