Membrane proteins are frequently present in crowded environments, which favour lateral association and, on occasions, two-dimensional crystallization. To better understand the non-specific lateral association of a membrane protein we have characterized the free energy landscape for the dimerization of a bacterial outer membrane protein, NanC, in a phospholipid bilayer membrane. NanC is a member of the KdgM-family of bacterial outer membrane proteins and is responsible for sialic acid transport in E. coli. Umbrella sampling and coarse-grained molecular dynamics were employed to calculate the potentials of mean force (PMF) for a variety of restrained relative orientations of two NanC proteins as the separation of their centres of mass was varied. We found the free energy of dimerization for NanC to be in the range of to . Differences in the depths of the PMFs for the various orientations are related to the shape of the proteins. This was quantified by calculating the lipid-inaccessible buried surface area of the proteins in the region around the minimum of each PMF. The depth of the potential well of the PMF was shown to depend approximately linearly on the buried surface area. We were able to resolve local minima in the restrained PMFs that would not be revealed using conventional umbrella sampling. In particular, these features reflected the local organization of the intervening lipids between the two interacting proteins. Through a comparison with the distribution of lipids around a single freely-diffusing NanC, we were able to predict the location of these restrained local minima for the orientational configuration in which they were most pronounced. Our ability to make this prediction highlights the important role that lipid organization plays in the association of two NanCs in a bilayer.
Cells are surrounded by selectively-permeable bilayer membranes, enabling the cell to control its internal environment. Embedded within these membranes are a variety of membrane proteins, many of which facilitate this environmental control and are integral to numerous metabolic processes. Their location within the membrane and their mutual association are controlled by many factors. We use molecular dynamics simulations to investigate the free energy of association for a pair of relatively simple membrane proteins. By doing so, we are able to characterize the effect that the geometrical properties of the protein have on their mutual association in a bilayer environment, showing that there is a correlation between the buried surface area of two proteins when in contact and the strength of their interaction. We also observe the effect of protein-lipid-protein interactions in this free energy characterization. Such interactions are related to the preferential distribution of lipids around proteins in the membrane.
Copyright: © 2014 Dunton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was undertaken using computing resources of the University of Oxford Advanced Research Computing (ARC) facility, the IRIDIS HPC facility provided by the e-Infrastructure South Centre for Innovation (EPSRC Grant ref EP/K000144/1, EP/K000136/1), and PRACE. TAD would like to acknowledge funding from EPSRC via the Life Sciences Interface Doctoral Training Centre. MSPS and JEG would like to acknowledge funding from BBSRC, the Wellcome Trust, and the EU (ScalaLife). JMO and DJG would like to acknowledge the EPSRC and Microsoft Research, Cambridge through grant EP/I017909/1 (www.2020science.net). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Cellular membranes not only separate the contents of a cell from its surroundings, they also play a key role in cell regulation and metabolism. Accounting for approximately a quarter of the coding regions of an organism's genome , membrane proteins control the transport of solutes between a cell and its surroundings, facilitate cellular movement, and regulate many aspects of cellular behaviour.
Gram-negative bacteria are surrounded by two membranes separated by a periplasmic layer. The outer membrane lipid bilayer is composed of phospholipids in the inner (i.e. periplasmic) leaflet, and of lipopolysaccharides in the outer leaflet. Within this membrane are many species of outer membrane proteins (OMPs), a class of integral membrane proteins whose secondary structures are almost exclusively . Many of these are porins (OmpC, OmpF, LamB, NanC, for example), through which small (approximately ) molecules can diffuse across the membrane. Porins provide a route for many antibiotics into bacterial cells and are potential vaccine targets .
Both in vivo and in vitro, membrane proteins are often present in a crowded environment. Thus, cell membranes generally have a high membrane area fraction (approximately 25% or greater) occupied by proteins . A similar degree of crowding may be found in membranes studied in vitro , . Such crowding may result in the clustering of proteins . Whilst the majority of discussion as to the nature of membrane protein cluster formation has focussed on lipid rafts , it should be noted that lateral interactions of crowded membrane proteins are a more general property –. In vitro, control of lateral association of a single membrane species in a highly crowded system may be used to induce two-dimensional crystallization .
Interactions within a crowded environment may lead to dynamic lateral interactions of membrane proteins, for example, those seen in recent time-resolved AFM studies of OmpF-containing membranes . In studying such interactions, one wishes to distinguish between specific oligomerization of membrane proteins (for example dimerization of transmembrane in glycophorin  and of in OMPLA ) and less specific interactions. Specific protein interactions are those in which the distributions of orientations of the oligomerized proteins are grouped almost exclusively into very few states (often only a single state). Less specific (or non-specific) interactions are those determined by other effects, such as (local) crowding, rather than purely due the specific interactions between residues on each of the proteins. In less specific oligomerization there may be some orientational dependence, but a more broad distribution of orientations is generally found among the oligomers. Benjamini and Smit found that it was important to determine the effect that non-specific interactions had on the crossing angle for pairs of before investigating the role of any specific interactions between the helices . It is therefore of interest to explore the energy landscape of lateral interactions of a relative 'featureless' OMP. NanC (Figure 1A) provides a good example of such a protein, as it is both structurally  and functionally  monomeric, whilst forming two dimensional crystals in DMPC lipid bilayers . NanC is member of the KdgM-family of bacterial outer membrane proteins, responsible for sialic acid transport in E. coli.
Figure 1. Atomistic and coarse-grained representations of NanC along with illustrations of the orientational combinations used in the PMF calculations.
(A) The atomistic structure of NanC is shown in the plane of the bilayer, which is perpendicular to the pore axis. (B–C) The coarse-grained NanC is shown in both the wide (B) and narrow (C) orientations, which are related by a rotation about the pore axis and with B being the equivalent orientation to A. The atoms/particles are represented by spheres with radii equal to their van der Waals radii. The atoms/particles from acidic residues shown in red, from basic residues in blue, from aromatic residues in yellow and from neutral residues in grey. (D–G) Four combinations of protein orientations as viewed from the extracellular side of the membrane. The traces of the NanC proteins illustrate their elliptical cross-sections. For each protein, the angle of orientation is measured between the line, which goes from the centre of mass of the blue protein through the centre of mass of the red protein, and the arrow, which goes from the protein's centre of mass through the of its isoleucine at residue 209. The orientational angle for each protein trace coloured blue is labelled and for each protein coloured red is labelled . The separation between the proteins' centres of mass is given by .
Experimental determination of the free energy of membrane protein dimerization in vitro has been used to characterize their properties in a membrane or membrane-like environment –. Characterization of the free energy landscape for membrane proteins gives us an insight into how the proteins will move and interact within the membrane and allows us to make predictions about their behaviour. There are many published examples of experimentally determined dimerization energies for membrane proteins and peptides –, , , but relatively few for proteins: one important example being the dimerization free energy of the phospholipase OMPLA, which was found to be in the region to .
Computer simulations provide a complement to both in vitro and in vivo experiments , enabling us to probe the microscopic interaction underlying membrane protein association. Molecular dynamics (MD) simulations have been used to explore a range of membrane proteins , in addition to related approaches such as Monte Carlo  and Brownian dynamics  simulations. In particular, simulations using a coarse-grained approximation  have been used to study dimerization of transmembrane domains  and of rhodopsin . In the latter case the simulations were also used to characterize large-scale organization of rhodopsin dimers into rows-of-dimers, as seen experimentally in disk membranes.
Many computational studies that explore free energy landscapes use the potential of mean force (PMF)  as a convenient description because it enables us to characterize a given reaction or transition as a function of a specific reaction coordinate (or set of coordinates). Not only does this enable us to characterize the free energy landscape as a function of the reaction coordinate (or coordinates), it also provides an opportunity subsequently to parameterize reduced models of complex systems using different simulation paradigms .
Calculations of PMFs for the association of membrane proteins have largely focussed on proteins. Thus, dimerization free energy landscapes for transmembrane have been calculated using MD with umbrella sampling ,  and with adaptive biasing force methods ; and also using Monte Carlo , and dissipative particle dynamics . These have yielded free energies of dimerization in the region of to . To date there has only been one computational study to calculate the association free energy of a membrane protein: the free energy of association for two OmpF trimers was estimated to be in the region of .
It has long been suggested that lipids play an important role in the interaction between proteins in a membrane , . For example, simulation studies have shown that hydrophobic mismatch may modulate the aggregation of proteins in the membrane , . It is therefore important that we capture the effects that lipids have on free energy landscapes if we are to understand membrane protein association in different bilayer environments.
In this paper we develop and apply a method for calculating the free energy of association for rotationally restrained proteins in a lipid bilayer. This allows us to resolve detailed structure in the (one-dimensional) PMFs, which reflect protein-lipid-protein interactions. We apply this method to characterize the association free energy of a coarse-grained model of NanC.
In order to characterize the proteins' free energy of association, we employed MD simulations of a coarse-grained model of NanC in a POPE bilayer to calculate the PMF , . Two orthogonal orientations of our coarse-grained NanC are shown in Figures 1B and C. It can be seen from these two views of the coarse-grained protein that, perpendicular to the pore axis, the protein is approximately elliptical in cross section, as it is wider in one direction (Figure 1B) than it is in the other orthogonal direction (Figure 1C). We calculated the PMF for four different orientational configurations of two NanC proteins, as shown in Figures 1D–G. These four configurations were chosen as examples of extremes of the possible contact regimes between the two proteins on association. In each of the configurations the proteins have either a wide (e.g. Figure 1B) or narrow face (e.g. Figure 1C) facing the other protein. These orientational configurations are categorized as: two configurations corresponding to maximal protein contact, where wide faces of both proteins face the other protein (shown in Figures 1D and E); one intermediate configuration, in which a narrow face of one protein faces a wide face of the other protein (shown in Figure 1F); and one configuration corresponding to minimal contact, where narrow faces of both proteins face the other protein (shown in Figure 1G). From these combinations of protein orientations we were able to investigate the differences between the PMFs for the various protein contact regimes.
The PMFs calculated for each of the four rotational combinations are shown in Figure 2. The PMFs were set to zero at an inter-protein separation of 8 nm, where the potentials have become approximately constant. The sampling methods, biasing potentials, rotational restraints and simulation details are given in the Methods section.
Figure 2. Potentials of mean force for four orientational configurations of the NanC proteins.
The four combinations are for angles of: ; ; ; and (in A, B, C, and D, respectively). The PMFs have depths of (A); (B); (C); and (D). In B, the minimum, labelled , and the two local minima, labelled and , correspond to inter-protein separations at which different numbers of lipids can optimally occupy the intervening region between the two proteins, as explained in the main text and illustrated in Figure 3.
We categorized the PMFs in Figure 2 by the depths of their potential well, which resulted in three categories of well depth. The first category contains the PMFs in Figures 2A and B, which both have depths of approximately occurring at inter-protein separations of approximately 3.2 nm. This first category corresponds to the orientational configurations of maximal contact, and , where wide faces of both proteins are brought into contact (shown in Figures 1D and E, respectively). It is interesting to note that the depths of the PMFs for these two parallel and anti-parallel orientational configurations are approximately the same. They are also similar in depth to the orientationally-unrestrained PMF calculated for this coarse-grained NanC system (shown in Figure S2), which has a depth of . This is much greater than the to calculated for the dimerization of OMPLA , the only experimental free energy for dimerization of an OMP, but as that was for a protein exhibiting specific oligomerization measured in detergent micelles, we would not expect a good agreement. The next category contains the PMF in Figure 2C, with a potential well depth of approximately occurring at a separation of approximately 3.5 nm. This corresponds to the intermediate orientational configuration in Figure 1F, where a wide face of one protein is brought into contact with a narrow face of the other. The decrease in the depth of the PMF indicates that the configurations with two wide faces in contact are more stable than this intermediate contact configuration, where . The third category contains the PMF shown in Figure 2D, which is the shallowest of the four PMFs with a potential well depth of approximately , occurring at an inter-protein separation of 3.5 nm. This PMF corresponds to the orientational configuration with minimal protein contact, where narrow faces of both proteins are brought into contact (shown in Figure 1G). This configuration is the least stable of the four configurations considered here. The correlation between the depth of the PMFs and the orientational configuration of the proteins suggests that the strength of the interaction may correlate with the overall extent of the resultant protein-protein interface.
As well as the restrained global minima (the global minima for the specific restrained orientations) of the potential wells in the PMFs of Figure 2, there are also multiple local minima, which occur at a variety of centre of mass separations. For example, the PMF in Figure 2B has a restrained global minimum (labelled ) and two higher-energy local minima (labelled and ), which we refer to as restrained metastable states. By fitting quadratic curves to the minima in Figure 2B we calculated their locations as 3.26 nm, 3.62 nm and 4.07 nm for , and , respectively.
The nature of the restrained global ( ) and local ( and ) minima is illustrated by the simulation snapshots shown in Figures 3A–C. These snapshots were taken from the simulation windows used to calculate the PMF in Figure 2B for an orientational configuration of . The snapshot shown in Figure 3A is from the umbrella sampling window in which the proteins were restrained with a separation of 3.3 nm, which is closest to the minimum at 3.26 nm in Figure 2B, labeled . We see that there is one lipid molecule between the two proteins at this restrained global minimum. It should be noted that this is the only lipid in between the two proteins; there is no equivalent lipid on the extracellular side of the membrane (the view from the other side of the membrane is shown in Supporting Information Figure S1), so the restrained global minimum configuration for this orientation has space for one lipid on the periplasmic side of the membrane. A snapshot from the umbrella sampling window with the proteins restrained with a separation of 3.6 nm is shown in Figure 3B, which is the window closest to the minimum at 3.62 nm, labelled in Figure 2B. We can see that there are two lipid molecules between the two proteins in this snapshot. The snapshot in Figure 3C is taken from the umbrella sampling window in which the proteins are restrained with a separation of 4.1 nm, which is the window closest to the minimum at 4.07 nm, labelled in Figure 2B, in which we see that three lipid molecules can occupy the space between the two proteins. These observations suggest that the existence of these restrained metastable states is a result of protein-lipid-protein interactions in this orientationally-restrained system.
Figure 3. Restrained metastable states in the PMFs can be explained in terms of the protein-lipid-protein interactions that result from the lipid ordering between the two proteins.
(A–C) Snapshots taken from umbrella sampling windows for the orientational configuration at inter-protein separations of 3.3 nm, 3.6 nm, and 4.1 nm, respectively. These snapshots correspond to the local minima in Figure 2B, which are labelled , and . The membrane is viewed from the periplasmic face, with the water and ions removed. The proteins are represented by traces through the particles of each residue, with one protein coloured blue and the other red. The lipid molecules are represented by spheres and are coloured by the lipid molecule, so that individual lipids can be identified. We see in the snapshot of state (A) that there is only one lipid between the two proteins, which is coloured purple. In the snapshot of state (B) there is room for two lipid molecules, coloured green and blue, to fit between the two proteins. In the snapshot of state (C) there is room for three lipids to fit between the two proteins, coloured in yellow, pink and orange. (D) Species density plot for the third coarse-grained particle in one of the tails for the lipids in the upper leaflet. The density is measured relative to the position of a freely diffusing NanC protein. The blue line is a projection of the particles onto the plane of the membrane. The arrow is the same arrow used throughout the text to show the protein's orientation and is drawn from the centre of mass through the of the isoleucine at residue 209. The dashed lines mark the angular region over which the mean lipid density (E) is measured and corresponds to the direction of the other protein in the orientational configuration . The mean is taken over both leaflets and all coarse-grained lipid particles. (F–H) The distribution is overlaid with a reversed version and aligned such that either one (F), two (G), or three (H) peaks occur in the region between the two proteins. The dashed line sections correspond to regions that are occupied by the proteins. The thick lines represent the overlaid lipid distributions around both proteins that correspond to the prediction of the lipid packed region between the two proteins. The vertical dotted lines indicate the edge of the reversed density plot. These edges correspond to the predicted position of the second protein and are located at 3.24 nm (F), 3.63 nm (G), and 4.02 nm (H).
To investigate the suggestion that these restrained metastable states were the result of the lipid ordering between the proteins, we calculated the lipid distribution around a freely diffusing NanC in a POPE bilayer. The distribution for a specific coarse-grained particle in the tail of all of the lipid molecules is shown in Figure 3D, where distinct annuli are visible, indicating regions of preferred occupation. We calculated the lipid distribution in a direction that corresponds to the direction of the other protein for the orientational configuration , indicated by the region between the dashed lines in Figure 3D. The average lipid distribution across both leaflets and all coarse-grained lipid particles in this direction is shown in Figure 3E, where again we can see there are preferred distances from the protein at which the lipids are observed. Further details of the averaging calculation are given in the Methods section.
We can use this directional lipid distribution to predict the separations at which the region between two proteins would be optimally packed by the lipid molecules. The alignment process is illustrated in Figures 3F–G and explained in the Methods section. For the minimum labelled in the PMF in Figure 2B, which occurs at a separation of 3.26 nm, we predict an optimal separation of 3.24 nm with one intervening lipid. For the first restrained metastable state labelled in Figure 2B, which occurs at a separation of 3.62 nm, we predict a separation of 3.63 nm with two intervening lipids. For the second restrained metastable state labelled in Figure 2B, which occurs at a separation of 4.07 nm, we predict a separation of 4.02 nm with three intervening lipids. Our predictions for the locations of the restrained metastable states are in close agreement with their location in the PMF. This supports our suggestion that the restrained metastable states observed in the PMFs are due to the protein-lipid-protein effects caused by the distribution of lipids between the two NanC proteins. For the other orientational configurations, the proteins have different faces facing the other protein and will therefore have a different optimal lipid distribution for each face. This may be one reason why the restrained local minima are better defined for and occur at regular intervals.
Such features are not usually observed in PMFs calculated with proteins that are free to rotate (for example, see Figure S2 for an orientationally-unrestrained PMF calculated for this coarse-grained NanC system). In the orientationally-unrestrained case the proteins would be able to rotate to alter the distance between their surfaces, provided they are not perfectly rotationally symmetric, so that the intervening region could be optimally packed with lipids without leaving any voids. However, for a system with rotationally restrained proteins, there is an optimal separation at which multiple lipid molecules can occupy the intervening space between the proteins.
Also observed in each of the PMFs is an energetic barrier, which occurs at an inter-protein separation of approximately 5.5 nm. Extending the arguments made above about the interaction of the two proteins individual lipid distributions, we can see that at distances greater than 2.5 nm in Figure 3E the fluctuations in lipid distribution have decayed to small oscillations around some constant average value, which indicates that these lipids are not as strongly influenced by the protein. From this argument we can think of this barrier as the point at which the lipids whose positions are strongly dependent on each protein begin to interact with one another, that is, there are lipids between the proteins that are affected by both of the proteins. We can think of this as the separation at which the annuli of lipids around each protein overlap with each other.
We wished to formally characterize the dependence of the PMF depth on the orientation of the proteins, which we suggested was related to the extent of protein contact. To do this we calculated the solvent accessible surface area (SASA) of the two proteins as a function of the separation of their centres of mass. For proteins with an approximately elliptical cross-section, we would expect the orientations with greater contact between the proteins to have a larger buried surface area. However, given the significance of the lipid effects that we identified above, it is important to corroborate this. Any features of the combined surfaces of the two proteins that would allow room for a lipid could have a large effect on the free energy.
The SASA is calculated using a spherical probe whose size determines the level of detail in the surface calculated for a specific set of atoms/particles. We used a probe with a radius of 0.47 nm, which is twice the radius of the coarse grained particles (0.235 nm) and should be a reasonable measure for the size of a lipid. We chose this size probe because it is the lipids that are the 'solvent' of interest when we bring two proteins together in a membrane. Further details are given in the Methods section.
For each of the four orientational configurations, Figure 4 shows the buried surface area as a function of distance from the minimum of their respective PMFs. We chose this measure since we wanted to remove the effect that the difference in protein radius has on the location of the minimum. In Figure 4 we see that there is a stratification of the buried surface areas in the region around the minima of the PMFs. As with the PMF depths in Figure 2, the buried surface areas can be divided into three categories. The buried surface area is largest for the orientations and , where the two wide protein faces are brought together. The next largest buried surface area around the minimum of the PMF is for the orientation , where one narrow face is brought into contact with one wide face. The smallest buried surface area around the minimum of the PMF is for the orientation , where two narrow faces are brought into contact. The correlation between the depth of the PMF and the buried surface area can be seen in the inset plot in Figure 4, in which these two quantities are plotted. We see that there is a negative correlation between the two quantities. For a protein orientation with a larger buried surface area, the minimum of the PMF is deeper.
Figure 4. Buried surface area of the protein complex as a function of distance from the minimum of the PMF.
The error bars correspond to . The inset figure shows the relative buried surface areas at the minimum of the respective PMFs as a function of the restrained global minimum PMF depth. The buried surface areas at the minimum of the PMFs were obtained from a straight line fit of the region from the minimum of the PMF.
We have characterized the free energy landscape of a pair of NanC proteins in a phospholipid bilayer. An interesting feature of these restrained free energy calculations is that certain restrained metastable states, which would usually not be seen, are now resolved. These local minima are associated with the ability of lipids to occupy the space between the two proteins at a given separation. Niemelä et al.  found that close to proteins, the lipids in a bilayer have reduced mobility, diffusing with the protein, and it is interactions involving these surrounding lipids upon protein association that we are observing here. Proteins' interactions with lipids have been shown to modulate local lipid formation , further demonstrating the important role of interactions involving both proteins and lipids in determining structures observed in the bilayer. PMFs for the dimerization of TM helices ,  and for other more complex proteins, including rhodopsin  and OmpF  have revealed similar features, suggesting that a role for lipids in the energetics of their interactions may be a general feature of membrane proteins. These features may not affect the kinetics of association, as we do not know if they present metastable barriers to association, but they will affect the dynamics of the system; the NanC proteins will need to negotiate the complex free energy landscape created by these protein-lipid-protein interactions if they are to reach an energetically stable state through oligomerization.
This result may also be compared with studies of membrane protein interactions using more approximate (and hence more general) models and DPD simulations . For example, such studies have suggested that changes in lipids may result in the modulation of mismatch-driven interactions of membrane proteins .
We identified a correlation between of the depth of the well in the free energy of association with the buried surface area at the interface of the two proteins. More generally, it has been suggested that oligomer stability of membrane proteins such as glycophorin A  and bacteriorhodopsin  may be correlated with the buried surface area at the interface. However, studies of the dimeric outer membrane protein OMPLA  failed to reveal such a correlation. This may reflect the role of lipids in OMPLA dimerization, confirming the need for detailed energy landscape calculations such as those presented herein.
Features of the methodology used in this work mean that care should be taken when interpreting the results. The treatment of solvents in the coarse-grained model is only approximate, so entropic contributions to solvation and lipidation/delipidation may not be captured as reliably as with a fully atomistic model. The nature of the coarse-grained model also does not enable us to separate out the contributions to the PMF due to energy and entropy, as is sometimes done using atomistic calculations. However, the observations we make here relating to the behaviour of lipids is mostly phenomenological and any quantitative observations are limited to relative comparisons between simulations of the same system. Furthermore, in choosing to look at a highly restrained system where the relative positions and orientations are restrained, we are also looking at the change in free energy along a narrow slice through configuration space. Although this path may be tightly defined, it is only by using such a highly restrained system that we are able to identify some previously unobserved behaviour, specifically the effect of protein-lipid-protein interactions on the free energy of protein dimerization. Such effects would usually be lost when averaging over a larger range of configurations.
The results presented here highlight some of the effects that contribute to the free energy of association for a bacterial outer membrane protein that undergoes non-specific oligomerization in a POPE bilayer. These processes will play a role in many protein-protein interactions, even those with some specific oligomerization modes, although in the latter case the non-specific interactions will likely be masked at close range by the specific interactions. We would expect the protein-lipid-protein interactions to be present in many membrane protein systems, as they seem to be determined by the underlying lipid-protein interactions. The PMF for the association of two OmpF trimers calculated by Casuso et al.  had a potential well that was approximately twice as deep as the ones we present here for NanC. However, the OmpF protein is much larger than NanC and the oligomerized proteins would therefore have a correspondingly larger buried surface compared to our NanC system.
Given the conclusions of this study, it will be of great interest to apply similar methods to those presented to calculate orientationally-dependent PMFs for a variety of other membrane proteins. Information obtained from PMFs, such as the orientational dependence of the free energy of association, are necessary for parameterizing yet coarser (i.e. more approximate) models (for example those of Yiannourakou et al. ), in order to enable simulation studies of the emergent properties of large, crowded and complex membrane models .
Umbrella sampling was used to obtain the PMF for each of the four orientational configurations while varying the inter-protein separation . The umbrella sampling was performed using simulation windows in which one protein was restrained at relative positions with the desired inter-protein separation. We chose this measure as our reaction coordinate because it is a natural choice for characterizing the separation of two proteins and it would also enable the PMFs to be used to parameterize larger scale models, as done by Yiannourakou et al. .
To calculate the PMF from these individual biased simulation windows we employed the weighed histogram analysis method (WHAM) . For the WHAM method to produce a converged PMF, we need to ensure that all points along the reaction coordinate are sufficiently sampled. This means that the histograms from the sampling of the reaction coordinate in each simulation window need to overlap with adjacent simulation windows and that these histograms are smooth. The effect of enforcing these requirements is that all points along the reaction coordinate are thoroughly sampled in multiple simulation windows. It is these considerations that determined our positioning of the umbrella sampling windows and the strength of the biasing potentials used in each.
For each orientational configuration, umbrella sampling windows were distributed at positions with varying inter-protein separation along a line connecting the centres of mass of the two proteins. The umbrella potential was applied to the centre of mass of each protein's particles, restraining them at relative positions with the desired centre of mass separation. The sampling windows were distributed at 0.1 nm intervals from an inter-protein separation of 2.8 nm to 8 nm. In each of these simulation windows we applied a harmonic umbrella potential with a force constant of . To improve the overlap of the histograms from adjacent simulation windows in the region of the local minima, which improved the resolution, additional simulation windows were used. These additional simulation windows were distributed at 0.05 nm intervals from a inter-protein separation of 2.8 nm to 4.5 nm, where the proteins were in close proximity. These closely separated windows had a stronger harmonic force constant of , in order that we could better resolve the barriers surrounding the restrained local minima.
The lipidation/delipidation of the protein-protein complex at close range is a slow process. It is important that we adequately sample both the lipidated and delipidated state. To do so, we also performed simulations in which that interface was manually delipidated, with the intervening lipids returned to the bulk of the bilayer, and the system re-equilibrated. Manually delipidated simulations were carried out for the orientational configurations , but were not required for as no persistent lipidated state was observed. Delipidated simulation windows were distributed from an inter-protein separation of 2.8 nm to 3.7 nm for , and to 4.0 nm for , separated by 0.5 nm in all cases and using the stronger position restraint of .
To analyse the free energy of association for specific relative orientations of NanC we had to ensure that we restrained their orientations in each simulation window, as well as their relative positions. This was achieved by applying a rotational potential to the particles of each protein. The rotational potential for each protein acted around a vector in the direction (approximately perpendicular to the plane of the membrane) through the protein's centre of mass. By applying a suitable rotational potential to the proteins, we were able to restrain their rotation without influencing the positional umbrella potential. Kutzner et al.  showed that we can virtually eliminate both the radial forces and forces parallel to the axis of rotation by using a restraining potential of the form (1)where is a unit vector parallel to the rotation axis; and are the current and reference positions of the particle, respectively; and are the current and reference positions of the centre of mass of the particles in each protein, respectively; is a rotation matrix, which describes the motion of the potential; is the force constant for the rotational potential, and is a small constant required to avoid a singularity at the axis of rotation.
The application of this potential to the particles of each protein results in a purely rotational force (a torque) about the proteins' centres of mass that acts in an approximately perpendicular direction to the plane of the membrane. This rotational potential is implemented by the enforced rotation feature of the GROMACS MD simulation package, which at each time-step applies an appropriate translational force to each of the restrained particles in order to create the desired torque , . The proteins were inserted into the membrane in the desired relative orientations for each configuration and so we wished to restrain their orientations, which was achieved by setting the rotation matrix, , equal to the identity matrix. The reference positions for the particles were taken from the initial protein structures at maximum separation. The force constant was set to , which kept rotational drift below , and the constant was set to . Kutzner et al. showed that values for greater than were shown to give good results for rotating the subdomain of ATPase using the same value of that we have used here .
To calculate the PMF of association for two NanC proteins we employed coarse-grained molecular dynamics simulations of a bilayer system. The system consisted of the two coarse-grained proteins embedded in a symmetric bilayer formed from 424 coarse-grained POPE lipid molecules. The bilayer was solvated and counter ions were added to neutralize the system. The coarse-grained force field we implemented was a modified version of the MARTINI forcefield , , in which approximately four heavy atoms were mapped to each coarse-grained particle. This mapping can be seen between Figure 1A and Figure 1B. All simulations were run using GROMACS v4.6 ScalaLife 2012 (available from http://www.scalalife.eu) . The simulations were performed under conditions of constant temperature (310 K) and pressure (1 bar) using a timestep of 40 fs. We have provided a GROMACS simulation configuration (mdp) file in the Supporting Information (Data S1) for a simulation window in which the protein restrained at relative positions corresponding to a centre of mass separation of 4 nm with a force constant of .
Each of the simulation windows were equilibrated for between and . The production simulations consisted of at least of simulation for each of the 0.1 nm separated simulation windows, where the applied umbrella potential force constant was . The length of simulation was increased if the PMF had not converged sufficiently. The convergence of each PMF was evaluated by comparing the PMFs obtained using non-intersecting subsets of production simulation data (see Figure S3). For each of the 0.05 nm separated simulation windows, where the larger force constant of was applied, of production simulations were performed. We also performed of production simulation for the manually delipidated simulation windows, which were separated by 0.05 nm. To combine the simulation data to obtain the PMFs we used the g_wham program, distributed with GROMACS , using a tolerance of .
In an attempt to predict the location of the restrained metastable states we performed a simulation of a single NanC protein freely diffusing in a POPE bilayer. This extended simulation consisted of a single coarse-grained NanC protein model (the same model used for the PMF calculations) embedded in a 25 nm square membrane constructed from coarse-grained POPE molecules. To analyse the lipid distribution around the single protein, we rotated each frame of the trajectory so that the NanC protein was aligned with its position at the start of the simulation. From this aligned trajectory we were able to calculate the position of the lipid particles in relation to the protein for the entire simulation. The particle density was calculated for a 6 nm square region around the NanC protein for each of the particles in the coarse-grained lipid molecules.
To calculate the protein density in a given direction, we calculated a linear projection of this two-dimensional density. For the case of the protein orientation configuration , the direction we are interested in is the same for both of the proteins, as they have the same face oriented toward the other protein. This direction is marked by the dashed lines in Figure 3D. In order to characterize the lipid particle density in this direction, we projected the two-dimensional density onto a series of 4 nm lines emanating from the protein's centre of mass, at regular angular intervals, within the region marked by the dashed lines. The dashed lines represent an angular window of and the individual projection lines were separated by .
To predict the location of the minimum and the local minima of Figure 2B, assuming the lipid behaviour corresponds to that shown in Figure 3D, we aligned the peaks of the mean lipid species plot (where the mean was taken across the linear projections for all coarse-grained lipid particles in both leaflets and is shown in Figure 3E) with those of the same plot overlaid with the x-axis reversed. For the case of a single intervening lipid we aligned the first peak with the first peak of the reversed plot (see Figure 3F). For the case of two intervening lipids we aligned the first peak with the second peak of the overlaid plot (see Figure 3G). Finally for the case of three intervening lipids, we aligned the first peak with the overlaid third peak and the second peak with the overlaid second peak (see Figure 3H).
To obtain the buried surface area of the proteins at various positions along the reaction coordinate, we analysed the surface area of the simulation windows with the higher translational restraining potential, , to enable the analysis of the surface area on a finer scale, using window separations of 0.05 nm instead of 0.1 nm. Using the higher force constant also ensured that the surface area was measured for a conformation that was sampled closer to the centre of the window; with the weaker force constant we would be measuring the surface area for conformations with separations that could differ significantly from the position of the window centre. All of the surface area calculations were carried out using the g_sas tool in GROMACS using of production simulation trajectory.
GROMACS configuration file. A typical configuration file (.mdp file) for one of the umbrella simulation windows where the proteins are restrained at relative positions corresponding to a centre of mass separation of 4 nm with a force constant of .
View of the extracellular leaflet. This is taken from the snapshot of the simulation shown in Figure 3A. Here we can see that the intervening region between the two proteins only contains the single lipid from the periplasmic leaflet, again shown in purple.
A PMF calculated for two NanC proteins in POPE bilayer, which have not been orientationally restrained. There are no observed local minima in this PMF. We calculated this PMF using the same simulation parameters as the orientationally-restrained PMFs, except the strength of the rotational potential, , was set to zero. Each of the simulations windows were equilibrated for and then production simulations were used to calculate this PMF. The PMF possesses many of the same features as the rotationally restrained versions, such as a small energy barrier around 5–6 nm and a deep potential well, but it is otherwise smoothly varying.
Rotationally restrained PMFs calculated using non-intersecting subsets of the simulation data, which are used to asses the convergence of the PMFs in Figure 2. The four orientational combinations are for proteins angles of: ; ; ; and (in A, B, C, and D, respectively). To evaluate the convergence of the rotationally-restrained PMFs presented in this paper we calculated two PMFs for each orientational configuration, each using half of the simulation data. The data used for each PMF constituted non-intersecting subsets of the total simulation data using to calculate the PMFs presented in Figure 2. From these PMFs we can see that the two versions agree to a high degree. The depths of the two PMFs calculated for each orientational combination are approximately the same and the local minima are observed at the same locations. There are a few regions where they do not overlap precisely, but these discrepancies do not affect the conclusions of the paper.
Conceived and designed the experiments: TAD JEG MSPS JMO. Performed the experiments: TAD JEG. Analyzed the data: TAD JEG. Contributed reagents/materials/analysis tools: TAD JEG. Wrote the paper: TAD JEG DJG MSPS JMO.
1. Nilsson J, Persson B, von Heijne G (2005) Comparative analysis of amino acid distributions in integral membrane proteins from 107 genomes. Proteins 60: 606–616.
2. Koebnik R, Locher KP, van Gelder P (2000) Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Molecular Microbiology 37: 239–253.
3. Nikaido H (2001) Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Seminars in Cell & Developmental Biology 12: 215–223.
4. Dupuy AD, Engelman DM (2008) Protein area occupancy at the center of the red blood cell membrane. Proceedings of the National Academy of Sciences of the United States of America 105: 2848–2852.
5. Le Brun AP, Holt SA, Shah DSH, Majkrzak CF, Lakey JH (2011) The structural orientation of antibody layers bound to engineered biosensor surfaces. Biomaterials 32: 3303–3311.
6. Casuso I, Khao J, Chami M, Paul-Gilloteaux P, Husain M, et al. (2012) Characterization of the motion of membrane proteins using high-speed atomic force microscopy. Nature Nanotechnology 7: 525–529.
7. Sieber JJ, Willig KI, Kutzner C, Gerding-Reimers C, Harke B, et al. (2007) Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317: 1072–1076.
8. Lingwood D, Simons K (2009) Lipid Rafts As a Membrane-Organizing Principle. Science 327: 46–50.
9. Mileykovskaya E, Dowhan W (2000) Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange. Journal of bacteriology 182: 1172–1175.
10. Mileykovskaya E, Dowhan W (2005) Role of membrane lipids in bacterial division-site selection. Current Opinion in Microbiology 8: 135–142.
11. Marguet D, Lenne PF, Rigneault H, He HT (2006) Dynamics in the plasma membrane: how to combine fluidity and order. The EMBO journal 25: 3446–3457.
12. Spector J, Zakharov S, Lill Y, Sharma O, Cramer WA, et al. (2010) Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex. Biophysical Journal 99: 3880–3886.
13. Pope CR, Unger VM (2012) Electron crystallography–the waking beauty of structural biology. Current Opinion in Structural Biology 22: 514–519.
14. Fleming KG, Engelman DM (2001) Specificity in transmembrane helix-helix interactions can define a hierarchy of stability for sequence variants. Proceedings of the National Academy of Sciences of the United States of America 98: 14340–14344.
15. Ebbie Tan AZ, Fleming KG (2008) Outer membrane phospholipase A dimer stability does not correlate to occluded surface area. Biochemistry 47: 12095–12103.
16. Benjamini A, Smit B (2012) Robust driving forces for transmembrane helix packing. Biophysical Journal 103: 1227–1235.
17. Wirth C, Condemine G, Boiteux C, Bernèche S, Schirmer T, et al. (2009) NanC crystal structure, a model for outer-membrane channels of the acidic sugar-specific KdgM porin family. Journal of Molecular Biology 394: 718–731.
18. Giri J, Tang JM, Wirth C, Peneff CM, Eisenberg B (2012) Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli. European Biophysics Journal 41: 259–271.
19. Signorell GA, Chami M, Condemine G, Schenk AD, Philippsen A, et al. (2007) Projection maps of three members of the KdgM outer membrane protein family. Journal of Structural Biology 160: 395–403.
20. Russ WP, Engelman DM (1999) TOXCAT: a measure of transmembrane helix association in a biological membrane. Proceedings of the National Academy of Sciences of the United States of America 96: 863–868.
21. Cristian L, Lear JD, DeGrado WF (2003) Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange. Protein Science 12: 1732–1740.
22. Doura AK, Fleming KG (2004) Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer. Journal of Molecular Biology 343: 1487–1497.
23. Stanley AM, Chuawong P, Hendrickson TL, Fleming KG (2006) Energetics of outer membrane phospholipase A (OMPLA) dimerization. Journal of Molecular Biology 358: 120–131.
24. Ebie AZ, Fleming KG (2007) Dimerization of the erythropoietin receptor transmembrane domain in micelles. Journal of Molecular Biology 366: 517–524.
25. MacKenzie KR, Fleming KG (2008) Association energetics of membrane spanning alpha-helices. Current Opinion in Structural Biology 18: 412–419.
26. Stansfeld PJ, Sansom MSP (2011) From coarse grained to atomistic: a serial multiscale approach to membrane protein simulations. Journal of Chemical Theory and Computation 7: 1157–1166.
27. Janosi L, Prakash A, Doxastakis M (2010) Lipid-modulated sequence-specific association of glycophorin A in membranes. Biophysical Journal 99: 284–292.
28. Cui M, Mezei M, Osman R (2008) Modeling dimerizations of transmembrane proteins using Brownian dynamics simulations. Journal of computer-aided molecular design 22: 553–561.
29. Monticelli L, Kandasamy SK, Periole X, Larson RG, Tieleman DP, et al. (2008) The MARTINI coarse-grained force field: extension to proteins. Journal of Chemical Theory and Computation 4: 819–834.
30. Sengupta D, Marrink SJ (2010) Lipid-mediated interactions tune the association of glycophorin A helix and its disruptive mutants in membranes. Physical Chemistry Chemical Physics 12: 12987–12996.
31. Periole X, Huber T, Marrink SJ, Sakmar TP (2007) G protein-coupled receptors self-assemble in dynamics simulations of model bilayers. Journal of the American Chemical Society 129: 10126–10132.
32. Roux B (1995) The calculation of the potential of mean force using computer simulations. Computer Physics Communications 91: 275–282.
33. Yiannourakou M, Marsella L, de Meyer FJM, Smit B (2010) Towards an understanding of membrane-mediated protein-protein interactions. Faraday Discussions 144: 359–367.
34. Chng CP, Tan SM (2011) Leukocyte integrin αLβ 2 transmembrane association dynamics revealed by coarse-grained molecular dynamics simulations. Proteins 79: 2203–2213.
35. Hênin J, Pohorille A, Chipot C (2005) Insights into the recognition and association of transmembrane alpha-helices. The free energy of alpha-helix dimerization in glycophorin A. Journal of the American Chemical Society 127: 8478–8484.
36. de Meyer FJM, Venturoli M, Smit B (2008) Molecular simulations of lipid-mediated protein-protein interactions. Biophysical Journal 95: 1851–1865.
37. Marcelja S (1976) Lipid-mediated protein interaction in membranes. Biochimica et biophysica acta 455: 1–7.
38. Killian JA (1998) Hydrophobic mismatch between proteins and lipids in membranes. Biochimica et biophysica acta 1376: 401–415.
39. Lagüe P, Zuckermann MJ, Roux B (2001) Lipid-mediated interactions between intrinsic membrane proteins: Dependence on protein size and lipid composition. Biophysical Journal 81: 276–284.
40. Parton DL, Klingelhoefer JW, Sansom MSP (2011) Aggregation of model membrane proteins, modulated by hydrophobic mismatch, membrane curvature, and protein class. Biophysical Journal 101: 691–699.
41. Bond PJ, Wee CL, Sansom MSP (2008) Coarse-grained molecular dynamics simulations of the energetics of helix insertion into a lipid bilayer. Biochemistry 47: 11321–11331.
42. Niemelä PS, Miettinen MS, Monticelli L, Hammaren H, Bjelkmar P, et al. (2010) Membrane proteins diffuse as dynamic complexes with lipids. Journal of the American Chemical Society 132: 7574–7575.
43. Domański J, Marrink SJ, Schäfer LV (2012) Transmembrane helices can induce domain formation in crowded model membranes. Biochimica et biophysica acta 1818: 984–994.
44. Periole X, Knepp AM, Sakmar TP, Marrink SJ, Huber T (2012) Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers. Journal of the American Chemical Society 134: 10959–10965.
45. de Meyer FJM, Rodgers JM, Willems TF, Smit B (2010) Molecular simulation of the effect of cholesterol on lipid-mediated protein-protein interactions. Biophysical Journal 99: 3629–3638.
46. Faham S, Yang D, Bare E, Yohannan S, Whitelegge JP, et al. (2004) Side-chain contributions to membrane protein structure and stability. Journal of Molecular Biology 335: 297–305.
47. Goose JE, Sansom MSP (2013) Reduced Lateral Mobility of Lipids and Proteins in Crowded Membranes. PLoS Computational Biology 9: e1003033.
48. Kumar S, Bouzida D, Swendsen RH, Kollman PA, Rosenberg JM (1992) The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. Journal of Computational Chemistry 13: 1011–1021.
49. Kutzner C, Czub J, Grubmüller H (2011) Keep it flexible: driving macromolecular rotary motions in atomistic simulations with GROMACS. Journal of Chemical Theory and Computation 7: 1381–1393.
50. Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. Journal of Chemical Theory and Computation 4: 435–447.
51. Hub JS, de Groot BL, van der Spoel D (2010) g_wham - a free weighted histogram analysis implementation including robust error and autocorrelation estimates. Journal of Chemical Theory and Computation 6: 3713–3720.
Is the Subject Area "Protein-lipid interactions" applicable to this article?


text:
Roko Blažević (* 10. März 2000 in Split) ist ein kroatischer Sänger. Er tritt als Sänger unter seinen Vornamen Roko auf.

Leben
Am 16. Februar 2019 gewann Roko den Wettbewerb Dora 2019. und vertrat somit Kroatien beim Eurovision Song Contest 2019 in Tel Aviv, Israel mit der englisch- und kroatischsprachigen Ballade The Dream. Unmittelbar danach stieg das Lied auf Platz 1 der kroatischen Charts ein. Am 16. Mai 2019 trat er im zweiten Halbfinale des ESC auf und erreichte letztlich Platz 14, wodurch er das Finale nicht erreichte.

Diskografie

Singles
 2018: Podsjećaš na ljubav
 2019: The Dream
 2019: Krila
 2020: Božić nam dolazi

Einzelnachweise

Popsänger
Interpret eines kroatischen Beitrags beim Eurovision Song Contest
Kroate
Geboren 2000
Mann

redpajama_set_name: RedPajamaWikipedia


text:
Invesco to Launch Four New Funds Tied to Flagship QQQ Tech ETF
IVZ 2.21%
is adding more Qs to its fund lineup, a wager by the world's fourth-biggest issuer of exchange-traded products that investors' love affair with technology stocks will continue.
The Atlanta-based asset manager will launch four new investment products tied to its flagship ETF, the
fund. At $134 billion, the Qs, as it is known across Wall Street, has grown to become one of the biggest exchange-traded products in the world and accounts for roughly 42% of Invesco's ETF assets.
QQQ offers investors exposure to the 100 biggest nonfinancial companies listed on Nasdaq Composite Index and has proved to be a huge draw since the throes of the 2008-09 financial crisis. The four new products look to build on that popularity and help Invesco compete in an industry dominated by
BlackRock Inc.,
BLK 0.54%
Vanguard Group and
State Street Corp.
"This is going to be an important part of our growth," said John Hoffman, Invesco's head of Americas, ETFs and indexes strategies. "We're trying to find all points in the ecosystem and make sure we're there to support it."
The new offerings have a retail bent and are rather simple as far as ETFs go. The Invesco Nasdaq 100 ETF (QQQM) will be a lower-cost version of QQQ designed specifically for buy-and-hold investors. Another, the Invesco Nasdaq 100 Index Fund (IVNQX) isn't an ETF at all. It is a mutual fund aimed at retirement-account investors, some of whom are restricted from owning ETFs.
The new suite is rounded out by a junior QQQ that features the second 100 biggest Nasdaq stocks (QQQJ) and an actively managed version that will hold the 25 stocks of the QQQ with the biggest growth prospects (QQQG).
Together, the new funds represent one of Invesco's biggest and most important product launches since a wave of closures following the purchases of OppenheimerFunds in 2019 and Guggenheim Investments' ETF business in 2018. Those deals added a host of new offerings that broadened Invesco's reach, especially overseas, as well as a glut of overlapping and overlooked products that Invesco closed.
The stakes remain high for Invesco. Shareholder activist Trian Fund Management has built up a roughly 10% position in the asset manager, along with a similar stake in smaller rival
Janus Henderson Group
JHG 1.34%
PLC, The Wall Street Journal reported earlier this month. Trian wants to force some consolidation within the industry, building a rival to the biggest asset managers in the world. An Invesco spokeswoman said the firm welcomed high-quality investors but declined to comment further.
Mr. Hoffman says he doesn't expect the four QQQ offshoots to stunt the growth of the flagship fund. QQQ is the second-most traded ETF in the U.S., making it highly liquid, an important component for big institutions that want to trade in and out of positions with few hitches.
And other than QQQM—the ETF that will be 5 basis points cheaper than QQQ—the other offerings serve distinct roles that would lend themselves to new money rather than transfers from QQQ.
The tech trade has been a clear winner over the past several years, as shares of those companies have been favored for their unrivaled growth and their resilience through economic dips, including the recent crisis brought on by the coronavirus pandemic.
Although the pandemic has crippled some industries, technology companies have been able to grow as people lean on the offerings of those companies to work, to stay connected with friends and families and to entertain themselves.
That has pushed QQQ up 39% so far this year, outpacing the broader Nasdaq index by roughly 6 percentage points. Over the past 10 years, the ETF is up nearly six times.
But tech stocks aren't impervious. The trade stumbled in September, dragging the broader stock market down with it amid myriad concerns ranging from ongoing shocks from the coronavirus to worries about whether the shares had climbed too far too quickly. The fund suffered some of its biggest outflows ever, shedding more than $6 billion on two separate days in September, according to FactSet.
Still, nearly $15 billion in net new money has flowed into QQQ this year, FactSet's data show.
Mr. Hoffman says QQQ and its offshoot funds are more than just a collection of technology companies. They include exposure to a number of sectors that use tech to their advantage, including biotechnology and retail.
"Innovation has never been so important," said Mr. Hoffman. "It couldn't be more front and center today [than] during a pandemic."
Write to Michael Wursthorn at [email protected]
Posted in new technology Tagged ETF, flagship, Funds, Invesco, launch, QQQ, Tech, tied

redpajama_set_name: RedPajamaCommonCrawl


Looks like it's definitely going to be Happy Holidays for Christmas and New Year. Snow depths for this time of year are the best they've been for several years. There's a great base and it looks like there'll be some sunshine over the next few days - a welcome respite from the arctic temperatures and low visibility skiing of last week. If you're heading out for Christmas you'll have a great time!
Snow Report 19th Dec 2017 Val d'Isere Tignes from tdc - the development centre on Vimeo.


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Home » Music » Queen Live Aid Anniversary: Roger Taylor reveals incredible moment when everything changed
Queen Live Aid Anniversary: Roger Taylor reveals incredible moment when everything changed
The blockbusting Bohemian Rhapsody film recreated the entire spine-tingling Live Aid setlist. And rightly so. Every second was pure magic and pure history being made, watched by millions around the world. The movie showed how the band realised more than most how to create 'stadium rock', music to engage a huge live audience with a clever hook, hand clap or call and response. But the band almost didn't take the stage at all, after years of divisions and doubts. So, it was particularly powerful when Taylor realised everything had suddenly changed.
The movie famously condensed some timelines and moved other events around. Some slammed the decision but Taylor and Brian May make no apologies for simply obeying the dictates of filmmaking.
Either way, the rifts within the band were very real at that time.
May recently told Mojo magazine how much the personal relationships between the band members had "suffered" through the early 1980s. Freddie Mercury, in particular, had disappeared into his own solo projects.
May said: "Freddie became estranged for a while… We definitely hesitated about doing Live Aid."
But the band also feared their lack of cohesion and loss of closeness might mean they were not able to deliver a proper performance.
May added: "The chances of making fools of ourselves were so big."
There was a lot riding on that one performance. Queen didn't just have something to prove to the world, they needed to prove it to themselves.
And then something remarkable happened as Freddie took the stage and the audience into the palm of his hand. The performance built to historic levels until Taylor looked out over the stadium at one special moment and just knew everything had changed.
Taylor told Mojo: " There was a point during We Are The Champions where the audience was swaying as one, like a field of wheat, and I thought, 'Oh, OK.'
"I went back to Freddie's later and at some point in the night said, 'You know what, Fred, I think we were really good.'
"It gave us a second wind."
LIVE AID JULY 13, 1985. QUEEN SETLIST: BOHEMIAN RHAPSODY, RADIO GA GA, AY-OH (Freddie call and response to the audience), HAMMER TO FALL, CRAZY LITTLE THING CALLED LOVE, WE WILL ROCK YOU, WE ARE THE CHAMPIONS.
Stephanie Niznik Dies: 'Everwood' & 'Star Trek' Actress Was 52
If You're Having A Crappy Day, Here Are 22 Tweets To Turn That Frown Upside Down


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Sustainable Product Design
London's 2012 Olympic Torch Isn't Green Enough
Bonnie Alter
Photo: london2012
The Olympic Torch is such an important symbol of the games. Passed from hand to hand by 8,000 torch bearers, it is an inspirational emblem for the host country.
London's Games organisers had hoped that theirs would be carbon-neutral, as an extra green and environmental gesture. But that has proved impossible to design. Where are all those clever kids when you need them?
Designed by Edward Barber and Jay Osgerby, it's a delicate but strong design.
It is 2 ft. 7 in. high, and made out of a lightweight aluminum alloy. It weighs 1.7 lb. so that young and old can carry it. The 8,000 holes (one for each of the torch bearers) make it light, allow people to see the flame, and also keep it cool. The gold colour is aesthetic as well as cooling for the flame.
The triangular design has been based on the number three: three Olympic values (respect, excellence and friendship), third Olympics for the UK (1908, 1948 and 2012), the vision for the Games (to combine work - sport, education and culture) and the three words of the Olympic motto (faster, higher, stronger). Pretty heady stuff.
Some have called it a vuvzela and others a cheese grate and golden spliff, but the designers say " We wanted to make the most of pioneering production technologies and to demonstrate the industrial excellence available in the UK - it's a Torch for our time."
The disappointment comes in the fuel. A butane and propane mix, such as used for gas stoves, will keep the flame burning. Ironically, it is sponsored by EDF who are proud to be "Britain's largest electricity generator, and we're committed to lower carbon energy production." However they couldn't make it carbon neutral, even though they have had since 2007 to figure it out. Their scientists tried to develop a carbon-neutral fuel using briquettes made from elephant grass which would burn with low emissions. Although these were as reliable and luminous as conventional gas-fueled torches, the scientists couldn't finish the design and engineering work.
In their defence, they have now come up with a "a bespoke low-carbon fuel based on a miscanthus briquette, which has been patented and could be used in domestic fires and garden torches." Maybe the next Olympic torch for Rio or Sochi can take advantage of this development.
More on Olympics
Touring the Olympic 2012 Site
Despite Huge Expense, London's Olympic Stadium Could Be Torn Down
London Olympic Stadium To Be World's Biggest Flatpack
Get your local, artisanal eyeglasses from Look Again
Scandi design + Portuguese cork: Buy your next phone case from 15:21
FREITAG stores are full of cardboard and chopped up old tarps. How ...


text:
He Wants To Choose His Costar
[Blind Gossip] Like many television shows, this one has seen cast members come and go over the years. As one female cast member transitions out, though, there is some very interesting maneuvering going on behind the scenes.
The male lead wants to pick the replacement. And his top choice is… the woman who nearly destroyed his marriage!
They had an affair a couple of years ago, and when his wife found out, she filed for divorce. Husband and wife eventually got back together, but his active lobbying for the other woman as his future costar certainly came as a surprise to us. His wife would probably be surprised as well.
His paramour is not the top choice of the producers, though, so it will be interesting to see if they give her a chance at his insistence. It will also be interesting to see if their behind-the-scenes chemistry translates into on-screen chemistry. We're going to pour ourselves a hot cup of coffee and watch how this one plays out today.
Male Lead:
Woman Leaving:
Woman's Possible Replacement:
It's The Today Show team!
Male Lead: Matt Lauer
Woman leaving: Ann Curry
Woman's Possible Replacement: TBA
Source: Blind Gossip
Matt Lauer just re-signed for close to $30 million a year (WOW!).
Ann Curry is searching for A new job on Monster.com.
Her replacement has not yet been announced. From TMZ:
Matt Lauer Re-Signs with 'Today'
Ann Curry on Chopping Block
Matt Lauer has signed a new, long-term deal with the "Today" show. He will become way richer than he already is, and we're told the next move will be to send Ann Curry packing.
As TMZ first reported, Lauer got close to his $30 million a year asking price, nearly doubling his $17 million a year current salary.
It's bad news for Ann Curry, because our sources say Lauer does not want her as his co-host, and it appears her fate is now sealed.
NBC sources say Ann's likely replacement will be Savannah Guthrie.
Did you catch the hints? Cup of coffee, today.
Iammeme was the first to get the first two right. Congrats to everyone who did, too! Good job!
Don't you feel special that you knew about this a week before everyone else did, just because you read Blind Gossip?
Well, the cat's out of the bag, so let's solve the second part of this.
Woman's Possible Replacement: Natalie Morales
Yes, Matt Lauer likely had an affair with Natalie Morales. It nearly wrecked both of their marriages. Even her husband doesn't deny it.
And – hold on to your hats – there are now rumors flying that they may have had a love child together! According to The National Enquirer:
IS IT TRUE WHAT THEY'RE SAYING ABOUT MATT LAUER?!
The rumors that just won't go away –MATT LAUER is caught up in an incredible shocking love child scandal!
On the heels of the "Today" host signing a fabulous $25 million-a-year contract with NBC, insiders at competing networks ignited nasty and unbelievable rumors that the 54-year-old married newsman fathered a child with his news anchor Natalie Morales.
"I heard that Matt had an extramarital affair with gorgeous Natalie, and he's the father of one of her kids," a rival network insider told The ENQUIRER. "Everyone's buzzing that the boy looks just like him."
Another industry source said: "The Matt Lauer love child scandal is the worst kept secret among the network morning shows.
"Everyone's talking about it – even at NBC. He's trying to get Ann Curry dumped as his co-anchor, and those loyal to her are also talking it up.
"Matt has enemies inside and outside the show's Studio 1A, and if he's got this giant skeleton in his closet, there are many who want to see it come out."
These incredible rumors about Matt are swirling just as the morning show wars have heated up to a fever pitch. "Today's" lost its lead over ABC's "Good Morning America", and CBS has relaunched its morning show with Gayle King as co-host, to stellar reviews.
But affable Matt is still keeping the "Today" show on top, while his enemies are busy trying to sink him with crazy stories.
The ENQUIRER discovered they have plenty of ammunition.
We revealed that Matt's roller-coaster marriage suffered a near-fatal blow in 2006 when his wife Annette – pregnant with the couple's third child – filed for divorce before withdrawing the legal papers a month later.
The divorce filing came while the unbelievable rumors were surfacing that Matt had gotten involved with Morales during the "Today" show's coverage of the 2006 Winter Olympics in Torino, Italy.
"They got very close and became extremely fond of one another," a source told The ENQUIRER. "They saw each other when they returned to the U.S."
Two years later, the rumors again began flying when Morales announced she was 12 weeks pregnant by husband Joe Rhodes.
"There was unbelievable talk that Matt could be the father of her baby!" revealed another show source.
Incredibly, when we gave Rhodes the opportunity to shoot down allegations that his wife was involved with Matt, he did not deny them to The ENQUIRER.
And Annette's fears about Matt's roving eye are well-grounded. Sources told The ENQUIRER that the balding broadcaster carried on with Jessica Thorman, a married Florida woman, until just months before his 1998 wedding to Annette.
Jessica's husband Zoltan Molnar accused Lauer of wrecking their marriage, stating: "He's not the goody-goody nice guy he looks like on TV!"
Published reports claim that Lauer is anxious to replace his "Today" show co-host Ann Curry, who assumed the position after Meredith Vieira left last year.
Sources say he wants a bigger personality beside him in the all-out ratings war that's seen his former TV partner Katie Couric recently sit in on "GMA" to boost its ratings with NBC tapping charismatic Sarah Palin to join Lauer as a guest co-host.
"The morning show competition has never been hotter and Matt is the reigning king," said the rival network source. "Everyone wants to see him get knocked off his pedestal and that's why these outrageous baby daddy rumors have resurfaced."
Morales, 39, remains part of the "Today" show team, reporting the news. She recently told an interviewer that she still manages to cook dinner every night for her investment banker husband Joe and their sons – Josh, 8, and Luke, 3.
Meanwhile, Lauer just bought 40 acres to build a horse farm down the road from his and Annette's $15 million mansion in the Hamptons.
Said the network source: "I think he's trying to keep Annette happy after years of causing her misery."
We've seen Natalie Morales' son, and we can't really tell if he resembles Matt. First of all, the kid has all his hair. And secondly, the kid gets along with Anne Curry just fine.
It will be interesting to see if Natalie still gets the co-host seat now that their little secret is out.
Congrats to everyone who got all three players right! IAmMeme was first!
BG Note: Yes, Matt Lauer did pick the replacement. Natalie Morales replaced Ann Curry as news anchor for Today in 2012.
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File Pic: HIV prevalence in young women reveals shocking figures
Over 700k young women living with HIV in Zimbabwe
11th April 2019 News Headlines
By Robert Tapfumaneyi
A staggering 740 000 young women in Zimbabwe are living with HIV, the third highest figures in the Sadc region, a UNAIDS HIV and Women report has revealed.
South Africa tops the list with 4.4 million followed by Mozambique at 1.3 million. The report describes a young women as one between the ages of 15-24.
The latest UNAIDS HIV and Women report released this week show that the reasons behind these figures vary and are complex, including the existence of high levels of transactional sex and age-disparate sexual relationships that are blamed for the HIV vulnerability of young women.
"An estimated 740,000 women were living with HIV in Zimbabwe in 2017.
"Gender inequality is present within relationships and marriages, and drives HIV infections," the report said.
"Although knowledge among young people is improving, only 37% of young women and 41% of young men have comprehensive and correct knowledge of HIV prevention.
"Only 46% of young women and 47% of young men have comprehensive knowledge about HIV, limiting their ability to take control of their sexual health."
The report added: "In addition, only 29% of adolescent women (aged 15-19), at high risk of HIV infection used a condom the last time they had sex, compared to 44% of their male counterparts."
UNAIDS said the low levels of condom use could be reflective of the fact that half of Sadc countries "impose age-restrictions on buying condoms."
The Report also said that in 2015, 17% of young women aged 15-19 in Zimbabwe reported having had sex with a man 10 years older in the past 12 months.
"This 'sugar-daddy' culture can contribute to an elevated risk of HIV for young women, as they are exposed to older men who may be more likely to have HIV or who hold the power in the relationship and determine condom use," said UNAIDS.
According to the report's findings, among young women, HIV prevalence increases with age. At least 2.7% of women aged 15-17 living with HIV, increasing to 13.9% for those in the 23-24 age group.
"Among young men, HIV prevalence holds steady at around 2.5% until the age of 23-24 when it increases to 6%," it said.
"However, as only 64% of young women (15-24) and 47.5% of young men have ever tested for HIV, prevalence among this group could be significantly higher."
Zimbabwe has one of the highest HIV prevalence rates in sub-Saharan Africa at 13.3%, with 1.3 million people living with HIV as at 2017.
Behind Zimbabwe in fourth is northern neighbour, Zambia with 670 000 young women living with HIV, followed by Malawi (630 000), Botswana (220 000) sits in sixth position with Angola (200 000) while Eswantini has 130 000 young women living with the deadly virus.


Background: Health literacy is the ability to obtain, process, and understand health information in order to take appropriate health actions. Low health literacy is associated with poor health knowledge and self-management of chronic disease, inadequate utilization of preventive services, and increased hospital admissions. The American Association of Colleges of Nursing recommends that nursing schools incorporate health literacy into curricula. Little, however, has been reported about what nursing students have learned and done about health literacy in clinical. This study explored undergraduate nursing students' experiences in caring for patients with low health literacy. Methods: A qualitative content analysis method was used to analyze 59 narratives written by undergraduate nursing students. Results: Three themes were uncovered: sensing low health literacy by behavioral cues, promoting health literacy with multiple strategies, and closing the health information loop with positive and negative feelings. Noncompliance, knowledge deficits, anxiety/concerns, and language barriers were behavioral cues indicating low health literacy, and these cues triggered the students' information support actions. Students promoted patient understanding and utilization of information by using many interventions: simplifying information, reinforcing information, giving written information, and demonstration/teach-back. Many students felt good about being able to help increase knowledge and self-care skills of their patients. Some were frustrated because they were unable to promote lifestyle modifications of the patients with complicated chronic diseases. Students, however, did not employ standardized tools to assess the health literacy of the patient or the patient's knowledge of specific diseases, nor did they assess readability of patient education materials or provide patient empowerment interventions to encourage active information-seeking and participation in self-care. Conclusions: Nursing students could identify behavioral cues suggestive of low health literacy and provide solutions to increase the patient's health literacy. To enhance student practice, nursing curricula, however, can integrate relevant health literacy assessment tools and empowerment interventions.


Motul is a world-class French company specialised in the formulation, production and distribution of high-tech engine lubricants as well as lubricants for industry via its Motul Tech activity.
Universally recognised for 165 years for the quality of its products and innovation capacity, Motul is also the specialist in synthetic lubricants. As early as 1971, Motul was the first lubricant manufacturer to pioneer the formulation of a 100% synthetic lubricant, the 300V.
Motul Racing Lab is a state-of-the-art mobile laboratory able to analyse the lubricant running in competitors' engines. In 2018, this free service is offered to all competitors for the first time in Goodwood Revival history.
Motul is also recognised as the specialist in synthetic lubricants. As early as 1971, Motul was the first lubricant manufacturer to pioneer the formulation of a 100% synthetic lubricant for automotive engines, the 300V lubricant, making use of Esters technology and issued from the aeronautical industry. Motul is supporting those teams in international competitions such as: 24 Hours of Le Mans (cars and motorcycles), FIA World Endurance Championship, IMSA WeatherTech SportsCar Championship, Super GT, Dakar , Blancpain Endurance Series, Dakar, Tour Auto, Le Mans Classic, MotoGP, World Superbike, IOM TT.
To celebrate the Revival partnership, Motul has created 1000 limited edition oil can, stay tuned to where you can get yours!


Description: Get ready to go shopping again with four of your favorite characters! And how else they could do it if not with a shopping session at the coolest fashion store in town - Urban Outfitters? Now shopping for fall outfits is even more fun than shopping for summer because fall fashion is all about layering shirts with cozy sweaters and fuzzy coats, while mixing them with the right pair of pants or with a skirt, chic knee-tall boots or with ankle wellies and some other cool fall accessories. So are you ready to get the fun started?


EDIT: Ok I just checked and the shock combo cannot be done if the sphere is a bit far away. It only happens in multiplayer.
If you don't get a sufficient answer let me know and I'll fire up my UT server. You can join and we can look at the logs to see what's up.
JackGriffin wrote: If you don't get a sufficient answer let me know and I'll fire up my UT server. You can join and we can look at the logs to see what's up.
I have these fake-lag issues with the vanilla version of UT too, I didn't have them before I installed the October version of Windows 10 from scratch.
EDIT: Also if I choose the shockrifle while playing a map from UnrealEd my inventory list in the HUD and the weapon itself disappear, I cannot shoot with it.
EDIT2: When I save a map with the editor, the camera is also saved.
You're too advanced an operator to ask these things but I will anyway in the interest of the people google searching this problem and finding your thread.
The fake-lag issues don't always happen, just sometimes, instead when I play the map I'm going to attach now with UnrealEd the inventory always disappears if I move the player view.
Have you tried using one of the latest OpenGL drivers?
Try MTR (or WinMTR if you're on windows), enter a server's IP and post results.
A couple of minutes should be enough.
In those moments you are getting lagged I'd try manually pinging the server and see if it's off the charts in that moment. I agree with Higor, once you exclude pretty much everything locally then it's got to be an ISP issue.
https://sites.google.com/site/ut99gotydownloads/home for a few examples.
MrLoathsome wrote: Have you tried using one of the latest OpenGL drivers?
Nothing changed, it cannot be a graphic driver problem at this point.
Higor wrote: Try MTR (or WinMTR if you're on windows), enter a server's IP and post results.
JackGriffin wrote: In those moments you are getting lagged I'd try manually pinging the server and see if it's off the charts in that moment. I agree with Higor, once you exclude pretty much everything locally then it's got to be an ISP issue.
Ping is the same that is displayed in the players list, I was on a server and the ping was just 42.
Chamberly wrote: Dual or multicore fixes?
I tried both, I tried using different versions of UT, including the multicore fixed one.
EDIT: Also the flag location is not aways updated, sometimes cappers still carry the flag even if they already capped and other times a flag is still at the base even if it actually isn't.
I wouldn't use the in-game ping for anything as a measure other than a baseline of comparison. At this point I'd consider something that runs all the time and keeps a log. This: https://www.paessler.com/ping-monitoring is way too much but it's in line with what you probably need.
How does normal speed tests/file downloading work for you? Is it consistent or does it also jump around? You might also try something like Hotspot Shield to run your game through a VPN. It's going to run slower for sure but you'll see if the lag spiking gets eliminated. It could be your ISP shaping traffic in high demand times.
IIRC Hitman had this same issue with his ISP when running his servers from his house and ended up moving the servers to another provider. I think the node he was on was just overloaded. That might be the case for you too.
JackGriffin wrote: I wouldn't use the in-game ping for anything as a measure other than a baseline of comparison. At this point I'd consider something that runs all the time and keeps a log. This: https://www.paessler.com/ping-monitoring is way too much but it's in line with what you probably need.
I just added a ZoneInfo, an island and some steel box decorations.
Last edited by PrinceOfFunky on Tue Jan 15, 2019 4:38 pm, edited 1 time in total.
Ah, I missed that part. Have you ran a serious spyware check? Something more robust that Malwarebytes? You could have picked up something and just be unaware. It's happened to all of us. I wouldn't think it was Windows10 on it's own unless you just have some weird setup that no one else has. Have you tested your RAM btw? Bad RAM can do odd things.
I'd hit a couple of the free rootkit scanners and maybe one or two online malware scanners just to be sure you don't have something hiding in there. Maybe even monitor your outbound traffic to watch for anything pinging out.
JackGriffin wrote: Ah, I missed that part. Have you ran a serious spyware check? Something more robust that Malwarebytes? You could have picked up something and just be unaware. It's happened to all of us. I wouldn't think it was Windows10 on it's own unless you just have some weird setup that no one else has. Have you tested your RAM btw? Bad RAM can do odd things.
I have a clean Windows 10 installation on VirtualBox, I use it to check if some programs are actually virus, maybe I could test UT99 on it through guest-additions.
JackGriffin wrote: Have you tested your RAM btw? Bad RAM can do odd things.
I would bet on this ... if your RAM needs a medic you'll never know what can happen.
Mouse freezing, stuff disappearing, BSoD because you hit "esc", buffer gets unloaded ... etc.


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text:
Book Review – Great City Maps
December 27, 2016 | By Chris Bruntlett
Author: DK Publishing (2016)
"A map is the greatest of all epic poems. Its lines and colors show the realization of great dreams." – Gilbert H. Grosvenor, Editor of National Geographic
For as long as humans have been building cities, they have been creating graphical representations of them, in a wide variety of styles, scales, and mediums. These maps have generally served as a design and record-keeping tool, but—retrospectively—they now tell a unique story of a moment in history; not just of the physical streets, buildings, parks, and public spaces that make up these urban environments, but of the values and traditions of the civilizations that shaped them.
Produced in partnership with the Smithsonian Institution, DK Publishing's Great City Maps is a carefully curated cross-section of these vital historical records, reproduced and annotated in a full-colour volume, ideal for your coffee table or office library.
In total, over 40 global cities are represented in over 70 different diagrams, organized into six categories: Ancient Cities (i.e. Rome, Jerusalem, and Mexico City), Medieval Trading Centres (i.e. London, Amsterdam, and Barcelona), Imperial Capitals (i.e. Vienna, Kyoto, and Moscow), Colonial Cities (i.e. Sydney, Cape Town, and Quebec City), Ideal Cities (i.e. Cairo, Paris, and Baghdad), and Megacities (i.e. Shanghai, New York, and Tokyo).
From the enormous and exhaustive plan of Ancient Rome carved onto 150 marble slabs on the side of a temple, to Hermann Bollmann's stunningly detailed axonometric projection of 1960s Midtown Manhattan, each of these graphic masterpieces is a snapshot of a region in transition, whether for better or for worse. Additional written historical, cultural, and economic context is provided, along with a visual tour of a number of noteworthy details that are enlarged and explained.
Each page will leave you with a newfound appreciation for the skill and imagination involved in mapmaking, as well as the endless days, months, and years needed to complete such a work of art. Consider, for example, the 1,500-sq. ft. mosaic of Byzantine-era Jerusalem painstakingly assembled on a church floor; or Sohei Nishino's six-foot tall collage of Tokyo, consisting of 4,000 hand-printed photos.
Great City Maps isn't just a study of cartography, but one of human history, and all of its triumphs and tragedies over the past 2,000 years. While our urban centres have served as important social and financial hubs over that period of time—setting the scene for amazing scientific, artistic, and technological accomplishments—this roids.co/ has also made them susceptible to devastation from disasters such as war, fire, famine, and disease. This fascinating book makes the compelling case that progress is worth the pain, and perhaps our single greatest achievement—the modern metropolis—is one worth documenting and celebrating.
For more information on Great City Maps, visit the DK website.
Chris Bruntlett is the co-founder of Modacity, a communications and marketing firm focused on inspiring healthier, happier, simpler forms of urban mobility through words, photography, and film.
Book Review | Mid-Century Modernism and the American Body: Race, Gender, and the Politics of Power in Design
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Andrew Rannells (center left) and Josh Gad (center right) lead THE BOOK OF MORMON cast. (Photo: Joan Marcus)
The Book of Mormon musical opens tonight on Broadway, but it seems it's already generating a considerable amount of buzz and becoming the "it" show of the spring Broadway season. Now that curiosity has cooled somewhat and preview performances are an interminability for that Spider-Man musical, it's time to take a closer look at a show that's earning its buzz.
As conceived by the creators of Comedy Central's long-running animated hit "South Park", Trey Parker and Matt Stone (who are not Mormons), the show is an offensively delightful musical comedy that attempts to satirize those Utah Mormons in their crisp white shirts and ties, the religious doctrines of the Book and The Church of Latter-Day Saints. The story follows two young Mormons who yearn to spread their message across the world. So they are sent packing from their Salt Lake City homebase on a far-flung mission to Uganda, of all places.
Advance praise has already been heaped by the likes of Entertainment Weekly, Rolling Stone and Vogue. Parker and Stone have also been making the TV publicity rounds with David Letterman and Jon Stewart. Apparently people are taking notice and creating great interest with positive word-of-mouth soundbites. Based on ticket demand alone, The Book of Mormon is selling very briskly and is on track to becoming the surprise hit of the season. A bawdy mormon musical, who knew?
Besides that, other new shows are worth mentioning in a bountiful spring lineup full of strong promise.
The drag divas of PRISCILLA QUEEN OF THE DESERT. (Photo: Joan Marcus)
For the musical front, my next pick would be Priscilla Queen of the Desert. Based on the 1994 Australian drag-queen film, Priscilla has now conquered New York after first doing Australia, London and Toronto. Other choices include Daniel Radcliffe in his singing debut in a new revival of How to Succeed in Business Without Really Trying and the musical caper Catch Me If You Can, another adaptation from the film that starred Tom Hanks and Leonardo DiCaprio, and a familiar Cole Porter musical, Anything Goes.
A hit London transfer from the National Theatre, War Horse leads my picks for plays. Based on the story by Michael Morpurgo and utilizing innovative puppetry design, it is told through a horse's perspective during WWI.
Several stars are enhancing quite a number of plays this season. Robin Williams headlines the war-themed comedy, Bengal Tiger at the Baghdad Zoo, as the titular tiger. Ben Stiller, Edie Falco and Jennifer Jason Leigh star in John Guare's The House of Blue Leaves, a revival concerning how a visit from the Pope in 1965 New York City can change a group of people's lives. An urgent and topical AIDS drama, The Normal Heart, comes back to New York next month, but in its first Broadway mounting. As written by Larry Kramer, this would be an important play to see that chronicles a man's struggle with an early epidemic of AIDS in the 1980's. Then there's The Motherfucker With the Hat. Starring Chris Rock and Bobby Cannavale, this is a foul-mouthed, R-rated comedy about going clean and sober. The title alone should give you an idea of what you're in for.
Categories: TheaterTags: Anything Goes, Ben Stiller, Bengal Tiger at the Baghdad Zoo, Bobby Cannavale, Book of Mormon, Broadway, Catch Me If You Can, Chris Rock, Daniel Radcliffe, Edie Falco, House of Blue Leaves, How to Succeed in Business Without Really Trying, Jennifer Jason Leigh, Larry Kramer, Matt Stone, Mormon, National Theatre, New York, Priscilla Queen of the Desert, Robin Williams, spring theater, The Motherfucker With the Hat, The Normal Hear, Trey Parker, War Horse
Newport Gets Jazzed Up
Ricky Martin Energizes in Propulsive Concert
Geronimo Sands
Geronimo Sands April 24, 2011 at 8:06 pm Reply
Beautiful website, Paul!
I am an old theatre friend of Rodney Hoel, who sent me to Culture Tourist.
How good to meet you this way. Keep up the great work.
Priscilla Beach Theatre


Citrix GoToTraining enables you to manage a stable and high-performance online training environment.
Description: Retrieves a list of trainings organized by the authenticated user.
Description: Retrieves all information about a scheduled training.
Description: Retrieves details of all registrants for a specific training.
Description: Registers one person, identified by a unique email address, for a training.
Description: Retrieves details of a specific registrant in a training.
Description: Triggers when a new training is created.
Description: Triggers when a registrant is added to a training.
Url link to join the meeting.
Unique identifier of the registrant.
Link to use by the registrant to join a training.
Link to use by the registrant to rsvp for a training.
Unique identifier of the training.
Where the training will take place.
Unique key of the training.