Water dynamics in rigid ionomer networks.

The dynamics of water within ionic polymer networks formed by sulfonated poly(phenylene) (SPP), as revealed by quasi-elastic neutron scattering (QENS), is presented. These polymers are distinguished from other ionic macromolecules by their rigidity and therefore in their network structure. QENS measurements as a function of temperature as the fraction of ionic groups and humidity were varied have shown that the polymer molecules are immobile while absorbed water molecules remain dynamic. The water molecules occupy multiple sites, either bound or loosely constrained, and bounce between the two. With increasing temperature and hydration levels, the system becomes more dynamic. Water molecules remain mobile even at subzero temperatures, illustrating the applicability of the SPP membrane for selective transport over a broad temperature range.

Effect of phosphorylation on the structural dynamics, thermal stability of human dopamine transporter: A simulation study using normal modes, molecular dynamics and Markov State Model.

The Human Dopamine Transporter (hDAT) plays an essential role in modulating the Influx/Efflux of dopamine, and it is involved in the mechanism of certain neurodegenerative diseases such as Parkinson's disease. Several studies have reported important states for Dopamine transport: outward-facing open state (OFo), the outward-facing closed state (OFc), the holo-occluded state closed (holo), and the inward-facing open state (IFo). Furthermore, experimental assays have shown that different phosphorylation conditions in hDAT can affect the rate of dopamine absorption. We present a protocol using hybrid simulation methods to study the conformational dynamics and stability of states of hDAT under different phosphorylation sites. With this protocol, we explored the conformational space of hDAT, identified the states, and evaluated the free energy differences and the transition probabilities between them in each of the phosphorylation cases. We also presented the conformational changes and correlated them with those described in the literature. There is a thesis/hypothesis that the phosphorylation condition corresponding to NP-333 system (where all sites Ser/Thr from residue 2 to 62 and 254 to 613 are phosphorylated, except residue 333) would decrease the rate of dopamine transport from the extracellular medium to the intracellular medium by hDAT as previously described in the literature by Lin et al., 2003. Our results corroborated this thesis/hypothesis and the data reported. It is probably due to the affectation/changes/alteration of the conformational dynamics of this system that makes the intermediate states more likely and makes it difficult to initial states associated with the uptake of dopamine in the extracellular medium, corroborating the experimental results. Furthermore, our results showed that just single phosphorylation/dephosphorylation could alter intrinsic protein motions affecting the sampling of one or more states necessary for dopamine transport. In this sense, the modification of phosphorylation influences protein movements and conformational preferences, affecting the stability of states and the transition between them and, therefore, the transport.

Unveiling mutation effects on the structural dynamics of the main protease from SARS-CoV-2 with hybrid simulation methods.

The main protease of SARS-CoV-2 (called Mpro or 3CLpro) is essential for processing polyproteins encoded by viral RNA. Several Mpro mutations were found in SARS-CoV-2 variants, which are related to higher transmissibility, pathogenicity, and resistance to neutralization antibodies. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. In this study, we used a hybrid simulation method to generate intermediate structures along the six lowest frequency normal modes and sample the conformational space and characterize the structural dynamics and global motions of WT SARS-CoV-2 Mpro and 48 mutations, including mutations found in P.1, B.1.1.7, B.1.351, B.1.525 and B.1.429+B.1.427 variants. We tried to contribute to the elucidation of the effects of mutation in the structural dynamics of SARS-CoV-2 Mpro. A machine learning analysis was performed following the investigation regarding the influence of the K90R, P99L, P108S, and N151D mutations on the dimeric interface assembling of the SARS-CoV-2 Mpro. The parameters allowed the selection of potential structurally stable dimers, which demonstrated that some single surface aa substitutions not located at the dimeric interface (K90R, P99L, P108S, and N151D) are able to induce significant quaternary changes. Furthermore, our results demonstrated, by a Quantum Mechanics method, the influence of SARS-CoV-2 Mpro mutations on the catalytic mechanism, confirming that only one of the chains of the WT and mutant SARS-CoV-2 Mpros are prone to cleave substrates. Finally, it was also possible to identify the aa residue F140 as an important factor related to the increasing enzymatic reactivity of a significant number of SARS-CoV-2 Mpro conformations generated by the normal modes-based simulations.

ABCG2/BCRP transport mechanism revealed through kinetically excited targeted molecular dynamics simulations.

ABCG2/BCRP is an ABC transporter that plays an important role in tissue protection by exporting endogenous substrates and xenobiotics. ABCG2 is of major interest due to its involvement in multidrug resistance (MDR), and understanding its complex efflux mechanism is essential to preventing MDR and drug-drug interactions (DDI). ABCG2 export is characterized by two major conformational transitions between inward- and outward-facing states, the structures of which have been resolved. Yet, the entire transport cycle has not been characterized to date. Our study bridges the gap between the two extreme conformations by studying connecting pathways. We developed an innovative approach to enhance molecular dynamics simulations, 'kinetically excited targeted molecular dynamics', and successfully simulated the transitions between inward- and outward-facing states in both directions and the transport of the endogenous substrate estrone 3-sulfate. We discovered an additional pocket between the two substrate-binding cavities and found that the presence of the substrate in the first cavity is essential to couple the movements between the nucleotide-binding and transmembrane domains. Our study shed new light on the complex efflux mechanism, and we provided transition pathways that can help to identify novel substrates and inhibitors of ABCG2 and probe new drug candidates for MDR and DDI.

Duloxetine 60 mg/day for the prevention of depressive recurrences: post hoc analyses from a recurrence prevention study.

OBJECTIVE: To assess the efficacy of duloxetine 60 mg/day in the prevention of depressive recurrence in patients with major depressive disorder (MDD). METHODS: Patients having at least three episodes of MDD in the past 5 years received open-label (OL) duloxetine 60-120 mg/day for up to 34 weeks. Patients meeting response criteria were then randomised to either duloxetine or placebo for up to 52 weeks of double-blind maintenance treatment. Only patients taking duloxetine 60 mg/day during the OL phase, and randomised to either duloxetine (remained on 60 mg/day dose) or placebo, were included in this post hoc analysis. The primary outcome measure was time to recurrence of a major depressive episode. The 17-item Hamilton Rating Scale for Depression (HAMD(17)) was used to evaluate depressive symptomatology. Global and physical functioning and pain were also assessed. Safety and tolerability were assessed via analysis of treatment-emergent adverse events (TEAEs), vital signs and weight. RESULTS: A total of 124 patients were randomised to duloxetine 60 mg/day (n = 64) or placebo (n = 60). Time to depressive recurrence was significantly longer in duloxetine-treated patients compared with placebo-treated patients (p = 0.001). During the double-blind maintenance phase, 31.7% of placebo-treated patients experienced a depressive recurrence compared with 12.5% of duloxetine-treated patients (p = 0.004). The HAMD(17) total score and most of its subscales as well as the Clinical Global Impression of Severity (CGI-S), significantly worsened in the placebo group compared with the duloxetine 60 mg/day group. There were no significant differences between treatment groups in TEAEs, discontinuations because of adverse events, vital signs or weight. CONCLUSIONS: Treatment with duloxetine 60 mg/day was associated with a longer time to depressive recurrence and a significantly lower recurrence rate compared with placebo.

Duloxetine in the treatment of major depressive disorder: a placebo- and paroxetine-controlled trial.

OBJECTIVE: Duloxetine doses of 80 and 120 mg/day were assessed for efficacy and safety in the treatment of major depressive disorder (MDD). METHODS: In this randomized, double-blind trial, patients age > or =18 meeting DSM-IV criteria for MDD were randomized to placebo (N=99), duloxetine 80 mg/day (N=93), duloxetine 120 mg/day (N=103), or paroxetine 20 mg/day (N=97). The primary outcome measure was mean change from baseline in the 17-item Hamilton rating scale for depression (HAMD(17)) total score after 8 weeks of treatment; a number of secondary efficacy measures also were assessed. Safety and tolerability were assessed via collection and analysis of treatment-emergent adverse events (TEAEs), vital signs, and weight. The Arizona sexual experiences scale was used to assess sexual functioning. Patients who had a > or =30% reduction from baseline in the HAMD(17) total score at the end of the acute phase entered a 6-month continuation phase where they remained on the same treatment as they had taken during the acute phase; efficacy and safety/tolerability outcomes were assessed during continuation treatment. RESULTS: More than 87% of patients completed the acute phase in each treatment group. Duloxetine-treated patients (both doses) showed significantly greater improvement (P<0.05) in the HAMD(17) total score at week 8 compared with placebo. Paroxetine was not significantly different from placebo (P=0.089) on mean change on the HAMD(17). Duloxetine 120 mg/day also showed significant improvement on most secondary efficacy measures (six of nine) compared with placebo while duloxetine 80 mg/day (three of nine) and paroxetine (three of nine) were significantly superior to placebo on fewer secondary measures. HAMD(17) mean change data from this study and an identical sister study were pooled as defined a priori for the purposes of performing a non-inferiority test versus paroxetine. Both duloxetine doses met statistical criteria for non-inferiority to paroxetine. TEAE reporting rates were low in all treatment groups and no deaths occurred in the acute or continuation phases. CONCLUSIONS: The efficacy of duloxetine at doses of 80 and 120 mg/day in the treatment of MDD was demonstrated. Tolerability, as measured by TEAEs, and safety were similar to paroxetine 20 mg/day and consistent with previous published data on duloxetine in the treatment of MDD.

Molecular orbital studies on the conformation of the phosphopantetheine moiety of coenzyme A.

Conformational study on phosphopantetheine shows that this compound has an intrinsic tendency to adopt a multitude of conformations which contain hydrogen bonds involving the sulphydryl, hydroxyl, carbonyl and amide groups. The sulphydryl group may form a hydrogen bond with the C(7') = 0 carbonyl group, the latter being also involved in hydrogen bonding with the N(4')-H GROUP. All these hydrogen bondings occur for different conformations around the backbone. The N(7')-H and C(4') = 0 groups are not involved in hydrogen bonding. It is also found that a strong interaction occurs between N(4')-H and 0-3' which is responsible for a rigid conformation around the C(3')-C(4') and C(3')-0(3') bonds. As far as the phosphate group is concerned the results show that this group may interact with the 0(3')-H hydroxyl group to form hydrogen-bonded rings of different sizes. A six-membered ring formed by hydrogen bonding between 0(3')-H and 0-1' appears more favorable than an eight-membered ring involving an anionic oxygen instead of an ester oxygen related to the phosphate group.

Molecular orbital calculations on the conformation of phosphodiesters. An extended correlation between the geometry and the conformation of the phosphate group.

An extension of our previous correlation (C.R. Acad. Sci. Paris, Ser. D (1973) 277, 2257; Biochim. Biophys. Acta (1974) 340, 299) between the geometry of the phosphate group and the conformation of phosphodiesters established by PCILO computations, shows that the probability of gauche-trans or trans-gauche conformations should become appreciable for low values (100-101) of the 33'-P-O5', angle and may even become predominant when the C-O-P angles have also a low value (congruent to 117 degrees). The results agree satisfactorily with available X-ray crystallographic data.

Molecular orbital studies on the conformations of 8-amino- and 8-dimethylaminoadenosine 5'-monophosphate.

The quantum mechanical PCILO method has been applied for the determination of conformational properties of 8-amino- and 8-dimethylaminoadenosine 5'-monophosphate. Contrary to other 8-substituted nucleotides the amino derivative shows a preference for an anti arrangement about the glycosidic bond. This conformation is stabilized by an intramolecular hydrogen bond between the purine and the exocyclic group. 8-dimethylamino-adenosine-5'-monophosphate adopts the syn conformation with slightly rotated dimethylamino group. There is, however, a local minimum for the anti form associated with the unusual value of chiCN = 300 degrees. This minimum is probably populated when the nucleotide is bound to lactate dehydrogenase apoenzyme. No particularly strong interactions are necessary for the stabilization of the anti form. The computations account satisfactorily for the available experimental data.

Theoretical study of the hydration of B-DNA.

A theoretical study of the hydration of the B-form of DNA has been carried out using empirical potential energy functions. In the first stage the hydration scheme of a model compound representing the B-DNA has been determined and the results have been shown to agree to a large extent with those of refined ab initio SCF computations. In the second stage, the stabilization energy due to the presence of water in the first hydration shell was computed by considering the hydrated helix as a supermolecule. The computations indicate appreciable stabilization. The different components contributing to the overall stabilization are determined and analysed.

A molecular orbital investigation of the conformation of transfer RNA.

The PCILO method has been used for a theoretical exploration of the conformational properties of tRNAPhe with respect to the phosphodiester torsion angles. The computations were based on the utilisation of the dinucleoside triphosphate model and took into account the different combinations of sugar puckers and different conformations about the C4'-C5' bond. The dependence of the (omega'-omega) conformational energy maps upon these factors was specified. A detailed comparison is carried out between the theoretical results and experimental data on the crystal structure of tRNAPhe produced by four different groups of investigators.

Motions in hemoglobin studied by normal mode analysis and energy minimization: evidence for the existence of tertiary T-like, quaternary R-like intermediate structures.

The normal mode analysis of human hemoglobin showed the presence in the deoxy T-state of one main preferential direction that brings the structure close to the R-state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate Tmin(d1), which is closer to R; the normal modes of Tmin(d1) indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to Tmin(d1), yielding Tmin(d2); then the procedure was repeated, yielding the intermediate structures Tmin(d3) and Tmin(d4). The structures Tmin(d2), Tmin(d3) and Tmin(d4) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, Tmin(d1), is different from the T and R structures, while the second set, Tmin(d2), Tmin(d3) and Tmin(d4), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha1 beta1 and alpha1 beta2 are R-like, and the alpha and beta heme environments are still T-like.

Intramolecular dielectric screening in proteins.

This paper investigates the microscopic mechanisms of charge screening by proteins. For this purpose, we introduce the generalized susceptibility of a protein in response to a point charge, which is a scalar quantity dependent on position within the protein. The contribution to the susceptibility from atomic polarizabilities, associated with electronic degrees of freedom, is found to be highly uniform. By contrast, that from dynamic dipolar relaxation, associated with nuclear degrees of freedom, varies greatly between different regions of the protein. We investigate the possible rôle of this variation in the activity of proteins that interact functionally with charged species, and we formulate and test the hypothesis that this variation is correlated to functional activity. Model calculations give encouraging support to this hypothesis. The protein's dielectric properties are represented by a standard model in which electronic relaxation is described by a set of atomic polarizabilities, and dipolar relaxation is treated as a perturbation to normal mode dynamics. The model yields the desired susceptibility in closed form. Its obvious limitations are discussed. It is applied to several test systems, and is compared to various continuum models. Four model alpha-helices are considered, three of which play a rôle in vivo in the binding of charged ligands. We show that the intramolecular screening, and its spatial variation, can indeed play a part in this binding. The electron transfer between ferri- and ferrocytochrome c is considered. The dielectric relaxation of each molecule, associated respectively with its oxidation or its reduction, is known to be directly related to the activation free energy for the electron transfer reaction. Our analysis of the dielectric susceptibility will thus permit an estimate of this activation free energy. We show that the relaxation of the atomic positions ("dipolar relaxation") contributes 1 kcal/mol to this activation free energy, and that the molecule achieves this low value by providing a low dipolar susceptibility throughout its central part. In this case, the spatial variation of the susceptibility has a clear functional rôle.

Analysis of the low-frequency normal modes of the R state of aspartate transcarbamylase and a comparison with the T state modes.

Aspartate transcarbamylase (ATCase) is a classic example of an allosteric enzyme. It catalyzes the conversion of aspartate to carbamyl aspartate, which is the first substrate in the biosynthesis of pyrimidines. Although ATCase is well characterized, both structurally and biochemically, little is known at the atomic level about the large amplitude motions that govern its T-->R quaternary transition. We present the results of calculations of the very-low-frequency normal modes of the CTP-ligated R state ATCase, and we compare them with the equivalent modes in the CTP-ligated T state ATCase. The large-amplitude, delocalized modes of frequencies below 4 cm-1 contribute a large fraction of the atomic fluctuations observed experimentally. They show some ability to drive the R-state structure towards the T-state structure, by promoting some of the quaternary structure rearrangements that take place during the allosteric process. Their potential role in the T-->R transition is quantified and compared with the role of the low-frequency modes of the T state in the quaternary rearrangement.

Analysis of the low frequency normal modes of the T-state of aspartate transcarbamylase.

Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli. It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally. As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist. In this work we have calculated the low frequency normal modes of the CTP-ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition. The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors. Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R-state structure is approached. The implication of these mode motions for the overall T-->R transition process is discussed.

Conformational dynamics and enzyme activity.

Conformational flexibility and structural fluctuations play an important role in enzyme activity. A great variety of internal motions ranging over different time scales and of different amplitudes are involved in the catalytic cycle. These different types of motions and their functional consequences are considered in the light of experimental data and theoretical analyses. The conformational changes upon substrate binding, and particularly the hinge-bending motion which occurs in enzymes made of two domains, are analyzed from several well documented examples. The conformational events accompanying the different steps of the catalytic cycle are discussed. The last section concerns the motions involved in the allosteric transition which regulates the enzyme activity.

Flexibility and folding of phosphoglycerate kinase.

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.

Treatment and outcomes for patients with depression who are partial responders to SSRI treatment: post-hoc analysis findings from the FINDER European observational study.

BACKGROUND: Remission is the goal in depression, but in practice many patients only experience a partial response to treatment. We sought to determine the prevalence, management and subsequent outcomes of partial responder patients. METHODS: Patients enrolled in the naturalistic Factors Influencing Depression Endpoints Research (FINDER) study with the Hospital Anxiety and Depression Scale depression subscale (HADS-D) score >10 at baseline who received only SSRI(s) between 0 and 3 months comprised the study cohort (n=1147). Patients were categorized as remitters, partial responders or non-responders at 3 months and then followed up at 6 months. RESULTS: At 3 months, 29.4% of the study population were considered non-responders, 27.6% were partial responders, and 39.3% were remitters. Most partial responders at 3 months remained on the same SSRI for the next 3 months. Of the 247 partial responders at 3 months and remained on the same SSRI(s) between 3 and 6 months, 10.9% met criteria for non-response at 6 months, 32.4% remained partial responders, and 56.3% achieved remission. Quality of life outcomes for the partial responders were significantly worse than those in remission (p<0.05). LIMITATIONS: FINDER was an observational study; the current analysis was conducted post-hoc. Multivariable methods were not applied and findings are primarily descriptive and exploratory. CONCLUSIONS: Partial response is common and patients in partial response have a poorer quality of life than those achieving remission. Despite this, the majority of partial responders continue to take the same SSRI. Our findings underscore the importance of continuing to strive for remission.