Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug–receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes, and activation of a G protein-coupled, inward-rectifying K^+ channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation–π interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed “rotamer switch” models. Interestingly, no comparable cation–π interaction was found at the aligning residue in the M2 receptor

Photodissociation dynamics of methyl iodide probed using femtosecond extreme ultraviolet photoelectron spectroscopy

Femtosecond pump-probe photoelectron spectroscopy measurements using an extreme ultraviolet probe have been made on the photodissociation dynamics of UV (269 nm) excited CH3I. The UV excitation leads to population of the 3Q0 state which rapidly dissociates. The dissociation is manifested as shifts in the measured photoelectron kinetic energy that map the extending C-I bond. The increased energy available in the XUV probe relative to a UV probe means the dynamics are followed over the chemically important region as far as C-I bond lengths of approximately 4 Å

Comparative Analysis of the Heptahelical Transmembrane Bundles of G Protein-Coupled Receptors

Background: G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices. Methodology/Principal Findings: We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands. Conclusions/Significance: Systematic analysis described here would be valuable for understanding of the rearrangement o

Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein

Photodissociation dynamics of CH3I probed via multiphoton ionisation photoelectron spectroscopy

The dissociation dynamics of CH3I is investigated on the red (269 nm) and blue (255 nm) side of the absorption maximum of the A-band. Using a multiphoton ionisation probe in a time-resolved photoelectron imaging experiment we observe very different dynamics at the two wavelengths, with significant differences in the measured lifetime and dynamic structure. The differences are explained in terms of changes in excitation cross-sections of the accessible 3Q0 and 1Q1 states and the subsequent dynamics upon each of them. The measurements support the existing literature on the rapid dissociation dynamics on the red side of the absorption maximum at 269 nm which is dominated by the dynamics along the 3Q0 state. At 255 nm we observe similar dynamics along the 3Q0 state but also a significant contribution from the 1Q1 state. The dynamics along the 1Q1 potential show a more complex structure in the photoelectron spectrum and a significantly increased lifetime, indicative of a more complex reaction pathway

Team climate, intention to leave and turnover among hospital employees: Prospective cohort study

<p>Abstract</p> <p>Background</p> <p>In hospitals, the costs of employee turnover are substantial and intentions to leave among staff may manifest as lowered performance. We examined whether team climate, as indicated by clear and shared goals, participation, task orientation and support for innovation, predicts intention to leave the job and actual turnover among hospital employees.</p> <p>Methods</p> <p>Prospective study with baseline and follow-up surveys (2–4 years apart). The participants were 6,441 (785 men, 5,656 women) hospital employees under the age of 55 at the time of follow-up survey. Logistic regression with generalized estimating equations was used as an analysis method to include both individual and work unit level predictors in the models.</p> <p>Results</p> <p>Among stayers with no intention to leave at baseline, lower self-reported team climate predicted higher likelihood of having intentions to leave at follow-up (odds ratio per 1 standard deviation decrease in team climate was 1.6, 95% confidence interval 1.4–1.8). Lower co-worker assessed team climate at follow-up was also association with such intentions (odds ratio 1.8, 95% confidence interval 1.4–2.4). Among all participants, the likelihood of actually quitting the job was higher for those with poor self-reported team climate at baseline. This association disappeared after adjustment for intention to leave at baseline suggesting that such intentions may explain the greater turnover rate among employees with low team climate.</p> <p>Conclusion</p> <p>Improving team climate may reduce intentions to leave and turnover among hospital employees.</p

Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques

Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally

Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation

The present study using zebrafish as a model explores the role of isotocin, a homolog of oxytocin, in controlling ion regulatory mechanisms. Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos. Whole-body Cl−, Ca2+, and Na+ contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants. In contrast, overexpression of isotocin caused an increase in ionocyte numbers. Isotocin morpholino caused significant suppression of foxi3a mRNA expression, while isotocin cRNA stimulated foxi3a mRNA expressions at the tail-bud stage of zebrafish embryos. The density of P63 (an epidermal stem cell marker)-positive cells was downregulated by isotocin morpholinos and was upregulated by isotocin cRNA. Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes

The type VII secretion system of <i>Staphylococcus aureus</i> secretes a nuclease toxin that targets competitor bacteria

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD(+) strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness