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Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.
Citation: Kentsis A, Gindin T, Mezei M, Osman R (2007) Calculation of the Free Energy and Cooperativity of Protein Folding. PLoS ONE 2(5): e446. doi:10.1371/journal.pone.0000446
Academic Editor: Hilal Lashuel, Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland
Received: January 17, 2007; Accepted: April 20, 2007; Published: May 16, 2007
Copyright: © 2007 Kentsis 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: National Institutes of Health Medical Scientist Training Program, Grant DK 43046.
Competing interests: The authors have declared that no competing interests exist.
The importance of accurately defining the molecular ensembles of proteins was recognized early by Levinthal, who concluded that folding of a random coil by way of a diffusive search of its combinatorially vast conformational space is incompatible with the biological energies and timescales of protein folding [1]. Consequently, either the conformational space of unfolded proteins deviates from that of a random coil, or the conformational search is not entirely diffusive, being guided by folding pathway(s), leading to proposals of hierarchical (thermodynamic) and framework (kinetic) folding models, respectively [2]–[5]. The extent to which these two fundamental mechanisms cooperate in the biological attainability of the folding search is unknown.
Structured unfolded states have been observed in a variety proteins [6]–[11]. However, it is unknown whether these macroscopically observed structures correspond to the conformations of individual residues, or to an average of microscopic configurational states that are composed of groups of residues. The former is consistent with the random, albeit conformationally biased (statistical) coil model of the unfolded state, and means that efficient folding is achieved largely by way of kinetic pathways. The latter is not, and implies that the unfolded state is thermodynamically pre-organized. Establishment of the extent of such pre-organization determines the relative contribution of the hierarchical (thermodynamic) and framework (kinetic) folding mechanisms, and is thus of major importance for understanding, predicting and designing biological macromolecules.
Study of this question has been made difficult by the spectroscopic limits of resolving microscopic ensemble sub-states that exist under the conditions of physiologic temperature, pressure, and hydration [12]. Such resolution is achievable theoretically by using molecular mechanics calculations, but is practically limited by the computational limits of simulating proteins in water under physiological conditions. These limits stem precisely from the dependence of conformational sampling of flexible solutes on the molecular properties of the solvent. Conventionally, these limitations are overcome through the implicit treatment of solvent effects, as in the approximation of Born [13]. However, its tested implementations do not appear to reproduce the thermodynamics and structures of natural proteins under physiologic conditions [14], though recently introduced algorithms appear to be more accurate [15]–[17].
Usage of Monte Carlo (MC) algorithms that utilize simultaneous changes of many conformational variables, such as loop torsion MC and replica exchange MC (REM), has shown promise in efficiently calculating convergent ensembles of proteins in aqueous solution [18]–[22]. However, application of loop torsion MC to protein folding depends on the analytical solutions of the loop closure problem, currently available for six polypeptide torsions [23]. REM or parallel tempering MC achieves changes of all conformational variables in aqueous solution through the use of global updates such as molecular dynamics (MD), but requires prohibitively large numbers of replicas in order to generate sufficient energy overlaps, as required by the Metropolis criterion [24], [25]. This Metropolis limit derives from the statistics of energy fluctuations, whereby the energy overlap between adjoining replicas required for efficient MC exchange scales as n−½, where n is the number of degrees of freedom, which are mostly of bulk water molecules. Recently, partial REM and REM with solute tempering (REST) have been developed to extend the Metropolis limit of REM for the simulations of protein folding in aqueous solution [26], [27]. Both do so by reducing the effective number of the degrees of freedom that contribute to the Metropolis energy criterion.
Here, we introduce another such variant, termed replica exchange MC with energy smoothing (REMS), that does so by manipulating the energy expression itself. We show that in spite of deforming the free energy surface to some extent, REMS yields apparently canonical free energy distributions in the energetic regime of biological systems. Consequently, we apply REMS to simulate the thermal folding of a small globular protein, the 20-residue Trp cage TC5b, under the near physiologic conditions of molecular hydration. We show that such an approach can be used for efficient and accurate calculation of protein stability ab initio, at least with respect to the folding of TC5b. And finally, by using self-consistent clustering and machine graph learning, we examine the origin of cooperativity and thermal stability of various structural motifs in this model protein. As a result, we offer a demonstration of the extent of thermodynamic and structural pre-organization of protein folding, important for understanding the mechanics of this phenomenon, with implications for a variety of problems, such as those that require calculations of free energies including structure prediction, genome annotation, and drug design.
Results and Discussion
Canonical molecular ensembles in water using REMS
TC5b is a small globular protein, consisting of several natural and redesigned structural motifs (Fig. 1). To generate a set of molecular ensembles of the thermal folding of TC5b, we equilibrated 32 replicas of TC5b in explicit water at 273–363 K, corresponding to the temperature range of experimentally measured thermal stability of TC5b [28]. This approach differs from earlier replica exchange simulations of TC5b [29]–[31], in particular by using periodic boundary conditions that are large enough (60×60×60 Å3) to accommodate a fully extended TC5b in explicit water, a 100 ps MD trajectory phase prior to replica exchange to achieve equilibration (Fig. 2A), and a 2 ps thermalization time during exchange to prevent quenching (Fig. 2B).
The structure is composed of an N-terminal α-helix with its α-helical/secondary Q5∶K8 salt bridge (red), type I β-turn S13-S14-G15 with its β-turn/tertiary D9∶R16 salt bridge (blue), and a hydrophobic core that includes both α-helical Y3∶W6 and tertiary W6∶P19 interactions (gold mesh).
A. Instantaneous potential energy (U) as a function of MD time during evolution of the 273 K replica in the canonical NVT ensemble prior to initiating REMS, demonstrating its equilibration, as reflected in the energetic stability during the last 50 ps. B. Instantaneous potential energy (U) as a function of MD time upon replica exchange from 276 to 273 K, demonstrating thermalization in less than 2 ps. C. Average potential energy 〈U〉 of 273 K replica as a function of energy smoothing time (ts). As ts approaches 2000 fs, the standard deviation of <U> approaches the fluctuation of the energy distribution in that time domain. At ts = 200 fs, energy-smoothed 〈U〉 of REMS is statistically indistinguishable from the instantaneous U used during conventional REM; double-sided p = 0.73. Bars represent ±1σ.
We used a smoothing time of 200 fs for the calculation of the Metropolis criterion during REMS (Methods), since the smoothed energy at this time shows small fluctuations, and most importantly, preserves approximately Boltzmann-weighted sampling. The difference between smoothed and near instant mean energies is less than 2.8 kcal/mol, and is not significantly different from that at shorter and near instant time intervals (Student's t-test p = 0.73; Fig. 2C), less than 10% of the total energy of the system. Usage of such energy smoothing leads to a distribution of and a mean potential energy of water (Fig. 3B) as well as temperature dependent heat capacity of water (Fig. 3C) which are statistically indistinguishable from those of exactly canonical simulations. On the other hand, usage of extremely long smoothing time of 600 fs leads to a gross underestimation of water's heat capacity, consistent with significant deviation from canonical sampling (Fig. 3C). Approximately canonical REMS with smoothing time of 200 fs leads to efficient replica exchange with mean exchange probabilities of about 30% (Fig. 4A); conventional REM of this system in explicit water being limited by Metropolis statistics of less than 1% (data not shown). Evolution of the calculated 32 ensembles for more than 4,000 exchanges with mean transition probabilities of about 30% means that the highest and lowest temperature replicas are exchanged on average more than 40 times, as confirmed by tracking the initial lowest and highest temperature replicas, containing the predominantly native and unfolded states, respectively, as they diffuse in temperature space in the course of the simulation (Fig. 4B). Consequently, the final simulation exceeds the apparent computational time constant of self-diffusion of TC5b by nearly three orders of magnitude (Fig. 4C), consistent with the simulation's convergence [21].
A. Histograms of potential energies (U) of different temperature replicas, demonstrating energy overlaps between adjoining temperature REMS replicas, as required for efficient MC exchange. B. Comparison of histograms of potential energies (U) of water ensembles at 273 K calculated using canonical MD (NVT) and REMS. Usage of REMS yields statistically indistinguishable mean energies and slightly increased energy fluctuations, as compared to those of canonical MD simulations, as shown by their normal fits (solid curves). C. Heat capacities at constant volume (CV) of pure water at different temperatures, as obtained experimentally (solid squares), and calculated using canonical MD (dotted diamonds) and REMS (dashed circles). Usage of REMS with extremely long smoothing time of 600 fs (REMS*, solid stars) leads to a significant underestimation of the heat capacity of water at low temperature. Sizes of symbols represent ±1σ.
A. Mean probabilities 〈P〉 of MC exchange between adjoining replicas xn and xm as a function of temperature, demonstrating that usage of REMS leads to efficient replica exchange. B. Exchanges of replicas in the temperature space, tracking the initial lowest (red dashed) and highest (blue solid) containing the predominantly native and unfolded states, respectively, as they diffuse in temperature space in the course of the simulation. C. Divergence of the normalized difference (Δ) of fraction of formed hydrophobic core W6∶P19 (closed squares), hydrophobic core Y3∶W6 (open circles), salt bridge D9∶R16 (closed stars), α-helical Y3∶L7 (solid circles) and the β-turn D9∶S14 (open squares) hydrogen bonds between initial and final structures as a function of replica exchange for the 363 K replica. These measure were chosen because their non-local nature should be most sensitive to initial configuration memory effects. The total length of REMS simulation exceeds the apparent computational time constant of self-diffusion by nearly three orders of magnitude.
Calculation of thermal stability of TC5b
In order to examine the origin of thermal stability of TC5b, we calculated the apparent stabilities of various conformational motifs of TC5b as a function of temperature (Fig. 5). Their choice was guided by the naturally occurring secondary and tertiary structural elements, as well as those that were specifically redesigned in TC5b [28] (Fig. 1). At 273 K, REMS calculated conformational ensemble of TC5b is largely folded, with nearly all molecules forming the N-terminal α-helix, the β-turn, the C-terminal polyprolyl helix (Fig. 5A), and tertiary and secondary hydrophobic cores (Fig. 5B). In contrast to the average NMR structure, the β-turn hydrogen bond and salt bridge, as well as the α-helical hydrogen bonds are largely (∼90%) but not persistently formed. On the other hand, the α-helical salt bridge is formed only in half of the ensemble (Fig. 5B), in agreement with the experimental observations of TC5b [28], [32]–[34].
A. Fraction of formed α-helix L2YIQWLK8 (dashed black), β-turn S14 (solid green), and polyprolyl helix P18 (dotted blue), as defined using self-consistent clustering and enumeration of their backbone dihedral angles. Note that P18 remains unchanged in its backbone conformation due to its definition in CHARMM. Individual α-helical residues have varying thermal stability, with the more N-terminal ones being less stable, consistent with the existence of α-helical fraying. B. Fraction of formed α-helical salt/secondary bridge Q5∶K8 (solid red), α-helical hydrogen bond Y3∶L7 (dotted red), β-turn/tertiary salt bridge D9∶R16 (solid blue), β-turn hydrogen bond D9∶S14 (dashed green), tertiary hydrophobic core W6∶P19 and Y3∶P19 (solid and dashed black), and secondary hydrophobic core Y3∶W6 (dashed red), as defined by using self-consistent clustering and enumeration of their distances. Note that the α-helical salt/secondary bridge is only partially formed at low temperature, even though the rest of the structure is nearly fully folded by other measures. Similarly, the secondary hydrophobic core Y3∶W6 persists even at high temperature, where the rest of the protein is largely unfolded by other measures. Importantly, substantial amount of residual native structure persists at high temperature. C. Fraction of formed mean α-helical structure (dashed black), mean β-turn structure (solid green), mean tertiary structure (solid black) in the REMS calculated ensembles, and native fraction measured experimentally using chemical shift dispersion (squares), as adapted from the first study of TC5b [28].
Stabilities of both local and non-local structural motifs exhibit an apparently sigmoid melting transition (Fig. 5). In particular, the N-terminal α-helix melts with an apparent melting temperature of 300–310 K, depending on the exact residue monitored, consistent with the presence of N-terminal fraying, wherein the helical residues closer to the terminus are less stable (Fig. 5A). Similarly, the β-turn melts with an apparent melting temperature of about 310 K (Fig. 5A), associated with the destabilization of the tertiary core and the β-turn/tertiary salt bridge (Fig. 5B). Calculation of the overall melting temperature of TC5b, by using mean ensemble conformational statistics, where all conformational motifs are equally weighted, yields an apparent value of approximately 310 K (Fig. 5C), in good agreement with the experimentally measured value of 315 K [28]. This finding differs from those of earlier replica exchange simulations of TC5b, which overestimated the apparent melting temperature by about 100 K, possibly because of continuum Born solvation [30], or constricted boundary conditions [29]. However, it is important to note that our study is limited by the use of a single (CHARMM) force field and initial (native) conditions, which may bias and limit sampling, respectively.
Unfolded state ensemble
Experimental studies of TC5b indicate a substantial amount of residual structure in the unfolded state ensemble at high temperature [28]. Our calculated high temperature ensemble also exhibits such structures, in particular possessing up to about 10% β-turn, 20% α-helical content, and 30% tertiary hydrophobic core (Fig. 5). Remarkably, nearly 60% of molecules in the high temperature ensemble contain the secondary hydrophobic core (Fig. 5B), in agreement with the experimental findings of such residual structure, as observed by using both NMR and fluorescence spectroscopies [28], [32]. This residual structure may arise from the persistence of various native-like conformations (isolated α-helical turn or hydrophobic core) in different molecules of the unfolded state ensemble that is otherwise non-native and heterogeneous. Conversely, this residual structure may be due to configurations of groups of conformations (associated α-helix and hydrophobic core) in an unfolded state ensemble that is relatively homogeneous with respect to these native-like configurations. Though indistinguishable macroscopically, these characteristically composed molecular ensembles diverge in the ways they affect protein folding and stability.
Graph manifold learning of the unfolded state ensemble
In order to discover the origin of residual structure at high temperature, we applied a graph-based approach designed to learn the natural coordinates of highly dimensioned data. By embedding the molecular ensemble in a graph based on geometric similarity, and projecting the individual structures onto a manifold that preserves nearest-neighbor geometric relations of this graph, we are able to distinguish globally organized configurations, termed mesostates, from groups of structures comprised of unrelated conformations (Methods). Indeed, the high temperature manifold is comprised of several such mesostates, including configurations of secondary structures such as the N-terminal α-helix and the β-turn, as well as more complex configurations that contain both the α-helix and the tertiary hydrophobic core, for example (Fig. 6). These configurations are not due to the use of the NMR structure as the starting configuration for REMS, as the latter's memory is lost after about 30 replica exchanges and the final ensemble is evolved for more than 4,000 exchanges (Fig. 4B&C). Instead, these configurations appear to pre-organize the unfolded state ensemble for folding by virtue of arranging individual interactions and conformations in the context of native-like mesostates. This pre-organization is likely inherent to the polypeptide sequence of TC5b, as suggested by energy minimization calculations of fragments of TC5b [35].
Mapping of the unfolded state ensemble, as calculated using the 363 K replica, onto the two top coordinates of its locally linear embedding space (open black circles), and the two top coordinates of its principal component projection (solid green circles). Principle component analysis fails to discern mesostate structure of the unfolded state ensemble, with the entire ensemble located near the origin of the PCA projection. On the other hand, displacement along the manifold from the origin of the LLE map coincides with the formation of native-like mesostates, containing: 1) α-helical/secondary salt bridge (red), 2) β-turn/tertiary salt bridge (blue), 3) α-helix and α-helical hydrophobic core, and 4) nearly native configurations with both the α-helix and the tertiary hydrophobic core.
Folding cooperativity and pre-organization
In order to estimate the extent of pre-organization of the thermal folding of TC5b by the residual structure of the high temperature ensemble, we calculated the apparent cooperativities of forming pairs of conformations into configurations, as expressed by the probabilities of forming these configurations conditional on the formation of their constituent conformations (Table 1). The conditional probabilities of forming these four-body interactions are related to the mesostate organization of the thermal folding reaction. Consistent with the presence of configurations containing the N-terminal α-helix in the high temperature ensemble (Fig. 6), the apparent conditional probability of forming both the α-helical hydrogen bond and the α-helical salt bridge exceeds the expected probability of forming this configuration assuming independence of its constituent conformations by more than a factor of 10 (Table 1). Similar effect is observed for the apparent cooperativities of formation of other configurations, involving both the secondary and tertiary hydrophobic cores and β-turn (Table 1). This surprising phenomenon is likely due to the thermodynamic coupling between the formation of individual native-like conformations and their organized configurations, such that native-like conformations are adopted essentially in the context of topologically native configurations.
Apparent coupling between local (conformational) and non-local (configurational) contacts has been noted earlier during the folding of Gō lattice polymers, where its origins were related to the details of the potential energy function defining the native state [36]–[38]. As the Gō protein model is supplemented with backbone interactions, local backbone conformations can lead to progressive non-local organization [39]. The finding of analogous conformational-configurational coupling during the folding of TC5b (Table 1), where on the other hand the folding process is defined by an atomic polypeptide in the context of a semi-empirical, classical force field (Methods), suggests that such coupling is inherent to the properties of the hydrated polypeptide itself.
The apparent cooperativity between forming concomitant α-helical and tertiary hydrophobic cores of TC5b exceeds the expected non-cooperative value by nearly a factor of 3 (Table 1). Because the formation of both α-helical and tertiary hydrophobic cores defines most of the native topology of TC5b (Fig. 1), this suggests that the residual unfolded state structure in the form of native-like configurations and mesostates at high temperature (Fig. 6) is responsible for most of the folding search. This phenomenon is similar to the pre-organization of α-helix formation in hydrated polyalanine [40], for which the microscopically pre-organized unfolded state contributes as much as half to the folding search [21], [41]. Altogether, the findings of such extensive pre-organization of both secondary structures as well as globular proteins suggest that the apparent biological efficiency of protein folding is due in large part to the thermodynamic pre-organization, as opposed to kinetic guidance. This pre-organization acts to reduce the conformational space available to the diffusive search of the unfolded state ensembles that are pre-ordered in configurational mesostates.
Graph manifold learning of folding mesostates
In order to assess how the thermal folding reaction can proceed by way of configurational mesostates, we examined the folding ensemble at the midpoint of its folding transition as comprised by the 310 K replica, by using graph manifold learning. At the folding midpoint, the unfolded and native state ensembles are equi-populated, and their inter-conversion defines all of the possible folding pathways [42]. Projections of individual configurations of the folding ensemble onto its LLE space map a star-shaped manifold, with multiple mesostates radiating from the origin of the projection (Fig. 7). Displacement along the M1 coordinate of the manifold coincides with the transformation between the native and unfolded structures, with configurations near the origin of the LLE map being partially native-like (Fig. 7). Displacements along the M2 and M3 coordinates coincide in part with the transformations of the α-helix and the β-turn, respectively, either in the context of native-like or unfolded topologies, depending on the particular location along the M1 coordinate (Fig. 7). The LLE mapping identifies a wide variety of folding mesostates, including those that possess a near native topology and α-helix but lack a native β-turn, those that lack the tertiary hydrophobic core and the native β-turn but retain the α-helix, as well as those that possess a near native β-turn and hydrophobic cores but lack the α-helix (Fig. 7). The existence of such mesostates explains the observed stabilities of their constituent conformational motifs (Fig. 5), as well as the apparent cooperativities of their configurations (Table 1). Combined with the star-shaped organization of the manifold of TC5b's folding ensemble, the variety of these folding mesostates suggest that the thermal folding reaction of TC5b is structurally heterogeneous. Though the folding of TC5b is pre-organized extensively by the unfolded state ensemble (Table 1&Fig. 6), this heterogeneity implies a relative diversity of available folding pathways, in agreement with experimental studies [43]. Determination of the exact subset(s) of folding mesostates that contribute to the kinetic transition state ensemble and the overall folding mechanism is an important direction of future work.
Mapping of TC5b's folding ensemble at the midpoint of its thermal transition, as calculated using the 310 K replica, onto the top three coordinates of its LLE manifold. Displacement along the M1 coordinate of the manifold coincides with the transformation of the 5) nearly native and 6) partially unfolded mesostates that lack the tertiary hydrophobic core and the native β-turn, but retain a frayed α-helix and the tertiary salt bridge. Displacement along the M2 coordinate coincides in part with the transformation of the α-helix from mesostate 7) that possesses a near native β-turn and hydrophobic cores and a non-α-helical but compact N-terminus, and mesostate 8) that lacks the native hydrophobic cores and has a non-native β-turn centered at K8 that is part of the N-terminal α-helix in the NMR structure. Displacement along the M3 coordinate coincides with the transformation of the β-turn, including mesostates 9) that have a near native β-turn and tertiary salt bridge but have an unfolded α-helix and hydrophobic cores, and 10) possess a near native topology and α-helix but lack a native β-turn.
Insofar as the free energy of flexible polymers can be described by a configurational partition function, our study shows that molecularly adapted variants of replica exchange, including REMS introduced here, can be used for the calculation of the free energy and cooperativity of protein folding ab initio. In addition, structural configurations and mesostates unknown a priori but adopted by the folding ensemble can be discovered and characterized by using graph manifold learning methods such as LLE. Our findings indicate that the thermal folding of a model globular protein, Trp cage TC5b, involves a structurally heterogeneous set of configurations and mesostates (Fig. 7). Some of these configurations persist in the molecular ensemble at high temperature (Fig. 6), concomitant with the pre-organization of TC5b's folding by such ordered unfolded state ensemble (Table 1). Combined with observations of thermodynamic pre-organization of polypeptide secondary structures [21], [41], these findings suggest that macromolecular modularity, as described by ensemble mesostates, likely plays an essential role in determining the structures and stabilities of biological macromolecules.
Furthermore, the successes and failures of current de novo protein design approaches likely reflect the significance of configurational organization of protein ensembles and the latter's contribution to protein stability, respectively [44]. Since TC5b's thermal stability and apparent folding cooperativity, two hallmark features of equilibrium folding of all natural proteins, are related to the residual structure of TC5b's unfolded state ensemble (Figs. 5&6), this suggests that the design of naturally stable proteins may be based on the structural preferences of unfolded polypeptides [45], as obtained computationally for example [46]. Indeed, TC5b has been re-designed recently by using just such an approach [47]. Application of advanced methods such as replica exchange Monte Carlo to sample the rugged energy spaces of proteins, and graph manifold learning to analyze the vast structural spaces of the molecular ensembles that constitute them, should prove useful for a variety of ab initio approaches to structure prediction, genome annotation, and drug design.
Molecular systems
To understand the origin of protein stability and cooperativity, we chose to examine a protein the folding of which is well characterized structurally, thermodynamically, and kinetically. The smallest such protein is the 20-residue Trp cage [28], TC5b (NLYIQWLKDGGPSSGRPPPS; Fig. 1), a derivative of the Gila monster extendin-4 that has been truncated and redesigned to include an N-terminal α-helix cap (N1), α-helical/secondary salt bridge (Q5∶K8), β-turn/tertiary salt bridge (D9∶R16), and optimized hydrophobic stack (Y3∶W6). In addition, TC5b contains a naturally occurring type I β-turn S13-S14-G15, type II polyproline helix P17-P18-P19, and a hydrophobic core containing both local secondary L2-Y3-I4 and non-local tertiary W6∶P18 and Y3∶P19 interactions.
NMR structure of TC5b (PDB code 1L2Y; model 1) was used as the starting configuration for our studies. The structure was solvated under periodic boundary conditions using a 60×60×60 Å3 cubic box of equilibrated TIP3 water, and energy minimized using the CHARMM27 potential energy function in the presence of one randomly placed chloride ion to yield electroneutrality [48], [49]. The resulting system was heated using molecular dynamics with a linear gradient of 20 K/ps and equilibrated in the isothermal-isobaric (NPT) ensemble at 273 K and 1 atm pressure for 100 ps, using the Leapfrog Verlet integrator with velocity rescaling, 2 fs integration time step, energy-conserving Nose-Hoover thermostat, SHAKE to constrain hydrogen atoms, center of mass updates to remove rotational and translational solute motion, and particle mesh Ewald (PME) summation to calculate electrostatic interactions, as implemented in CHARMM. Because these calculations were already in progress, we did not use the subsequently introduced CHARMM backbone dihedral parameter correction [50]. Upon equilibration, systems were 60.7×60.7×60.7 Å3 in volume, containing a total of 21,640 atoms and 7,112 water molecules. Such size and equilibration was necessary to thermalize and unfold this protein (see below). This system was used as the initial state for molecular dynamics equilibrations in the canonical (NVT) ensemble for 100 ps at mean temperatures of 273+3n K, where n = (0, 31).
Replica exchange
For REM, we utilized the MMTSB Tool Set, a recently developed collection of Perl scripts that interface with CHARMM [51]. Thirty two replicas were prepared as described above, thermalized at temperatures that were spaced between 273 and 363 K, a range chosen based on the experimentally observed thermostability of TC5b [28]. Each replica was simulated independently in the canonical ensemble under periodic boundary conditions using Leapfrog Verlet molecular dynamics with velocity rescaling, 2 fs integration time step, Nose-Hoover thermostat, SHAKE constraint, and PME electrostatics. Every 2 ps, an exchange between replicas n and m neighboring in temperature was attempted using the energy smoothed Metropolis criterion: P (xn | xm) = 1 if ΔE ≤ 0 and P (xn | xm) = exp(–ΔE) if ΔE>0, where P is the probability of exchange, ΔE = βn 〈Un〉ts–βm 〈Um〉ts, β = 1/kBT, U is potential energy, and ts is the MD smoothing time preceding the exchange over which the energies of the instantaneous configurations xn and xm are box averaged. Value of ts was tuned interactively to produce mean exchange acceptance rates of about 30%, while preserving approximately Bolzmann sampling, with ts of 200 fs used to generate the results described below. In the limit where the smoothing time is very long and the distribution of energy approaches the fluctuations in that time domain, e.g., ts>600 fs (Fig. 2C), REMS is expected to produce ensembles with significantly reduced energy differences among states, allowing transitions between states that would otherwise by very different in their energies.
Upon each exchange of replicas neighboring in temperature, another exchange using the new pairs of neighboring replicas was attempted in order to maximize the tempering effect and the movement of replicas across the sampled temperature range. Upon a completed exchange, velocities of the exchanged configurations were rescaled to the new temperatures, another exchange was attempted 2 ps later, and the entire REMS simulation was produced for a total of 4,710 exchanges, while discarding 100 initial exchanges, corresponding to more than 0.3 µs of aggregate MD time, and sampling more than 150 million configurations.
Energy smoothing of REMS is equivalent to introducing an error into the calculation of the Metropolis criterion, and consequently produces non-stationary distributions of Markov chains of configurations. Though different in origin, this feature of REMS is analogous to the lack of stationary distributions produced by other tempering methods such as variants of Jump-walking (J-walking), where the conventional MC walker is allowed large transitions sampled from a different temperature ensemble, yielding generally non-stationary distributions of states [52], [53]. Nevertheless, it can be shown that approximately canonical distributions of states can be generated by using tempering MC methods such as J-walking when the jumping frequency is low compared to the total length of the MC walker, e.g., when the deformation of the free energy of the system is small relative to the differences in energy of major ensemble states [54], [55].
In order to evaluate the suitability of REMS to actually recover canonical energy distributions, we calculated the constant volume heat capacity of pure water: Cv = (〈U2〉–〈U〉2)/kBT2. Because heat capacity reports squares of energy fluctuations, it is an extremely sensitive measure of the equipartition of energy that characterizes canonical ensembles. For this purpose, we used a 20×20×20 Å3 box of equilibrated TIP3 water under periodic boundary conditions, simulated using MD in the canonical ensemble for 1 ns, using MD protocol as described above, at four different temperatures: 273, 285, 299, and 313 K. We carried out a REMS simulation of the same system, using replicas at 273, 285, 299, and 313 K, simulated for 1,000 exchanges attempted every 1 ps with ts of 200 fs, corresponding to aggregate MD time of 1 ns, equal to that of simulations using canonical MD without REMS. As can be seen from Fig. 3, usage of the energy smoothed Metropolis criterion does not lead to any significant distortions of the mean energy of water under near physiologic conditions of temperature and pressure, as seen from the comparison of the results of MD NVT and REMS simulations. More importantly, no statistically significant differences exist between the constant volume heat capacities of water calculated using exactly canonical MD NVT and approximately canonical REMS, both of which are in good agreement with the experimentally measured values (Fig. 3) [48].
To evaluate the computational efficiency of REMS, we calculated mean transition probabilities of exchanging pairs of replicas adjoining in temperature during the course of the simulation of the thermal folding of TC5b. As can be seen from Fig. 4, usage of REMS improves the otherwise system size-limited parallel tempering MC, yielding mean exchange acceptance ratios of about 30%, similar to traditional MC transition probabilities. To evaluate sampling efficiency, we calculated the evolution of the apparent self-diffusion coefficient Δ0−N = (f(0) − f(N))/f(final), as a function of simulation length with respect to the number of replica exchanges N, where f is a phase space variable, such as the fraction of the native hydrophobic core of TC5b. If the sampling of phase space is ergodic, Δ0−N decays to one at long N. This is a necessary but insufficient condition of ergodicity, since it depends on the choice of initial and final conditions. Due to the requirement of carrying out multiple independent simulations, we are unable to evaluate ergodicity directly [21].
Microscopic analysis and clustering
In the analysis of structures of calculated ensembles, we use the term conformation to refer to geometries of individual interactions, and configuration to refer to molecular geometries of groups of interactions. Although canonical structures, such as α-helices and β-turns, have defined regular geometries, conformations in solution at ambient temperature exhibit considerable plasticity. Thus, we utilized a self-consistent method for defining conformational basins using a stepwise optimal clustering algorithm based on a self-organizing neural net, as implemented in ART-2 by Brooks and coworkers [56]. Briefly, the cluster assignments of structural variables extracted from simulation ensembles were minimized subject to a constraint on a cluster radius, such that no member of a cluster was farther than a specified distance from the cluster center. Because the convergence of such minimizations is sensitive to initial conditions, we tested the robustness of assignments to conformational basins by recalculating cluster assignments using reshuffled trajectories (data not shown). Cluster occurrences and probabilities of sampling of conformational basins as defined in this manner were calculated using a set of home-built programs, available upon request.
In this manner, we examined the formation of the N-terminal α-helix by clustering (Φ,ψ) dihedral angles of the L2YIQWLK9 polypeptide backbone and intrahelical hydrogen bond distances between backbone amide hydrogens and carbonyl oxygens, formation of the α-helical/secondary Q5∶K8 salt bridge by clustering the distance between side chain Q carboxamide oxygen and K amine nitrogen, formation of the β-turn by clustering (Φ,ψ) dihedral angles of S14 and the hydrogen bond distance between backbone D9 carbonyl oxygen and side chain S14 hydroxyl hydrogen, formation of the β-turn/tertiary salt bridge D9∶R16 by clustering the distance between side chain D carboxylate carbon and R guanidino nitrogen, formation of the polyproline helix by clustering (Φ,ψ,ω) dihedral angles of P18, and lastly, formation of the hydrophobic core by clustering contact distances among side chain Y3 phenol carbon ζ, W6 indole carbon δ, and P19 imido carbon δ. For all conformational variables, probabilities of forming native conformations were calculated by using clusters with near native centroids, as referenced to the NMR structure of TC5b.
Folding manifold learning
Because probabilities of forming structural configurations, such as folding intermediates, cannot be derived from conformational probabilities a priori, we examined their occurrence by direct enumeration of conditional probabilities of forming pairs of conformations. Apparent cooperativities of forming pairs of native interactions were calculated by using Ppair = P (i+j|i ; j), the probability of forming both interactions i and j under the condition that either i or j is formed.
In order to discover configurations that involve more than four-body interactions described above, we applied non-linear graph manifold learning techniques. Conventionally, study of high dimensional data such as atomic protein folding trajectories has been done using linear methods such as principal component analysis (PCA). PCA works by computing linear projections of greatest variance from the top eigenvectors of the data covariance matrix, thereby preserving the covariance structure of the data. However, because the global structure of high dimensional data is not necessarily linear, low dimensional linear principal components fail to capture this structure adequately (Fig. 6) [57]. Recently, graph based methods, including locally linear embedding (LLE), have been developed to preserve data neighbor relationships without enforcing global linearity [58]. Simply put, such methods provide compact representations of complex data without imposing artificial constraints.
Our LLE input data set was dimensioned using the Cartesian coordinates of heavy atoms of TC5b (154 atoms × 3 (x,y,z) = 462 dimensions), and included 2,355 configurations sampled from the 363 K replica to model the unfolded state ensemble, or from the 310 K replica to model the folding ensemble. All coordinates were centered and oriented with respect to the NMR structure of TC5b (PDB model 1, see Methods) in order to simplify the calculated manifolds, though this procedure is not required, in contrast to PCA [59], [60]. LLE was carried out by calculating Euclidean distances between individual configurations, as defined by the Cartesian coordinates of their heavy atoms, and constructing nearest neighbor graphs using k-means clustering to define nodes of k-nearest neighbors. For the results shown below, we used k of 18. Varying k between 12 and 20 produced no qualitative differences in resulting manifolds (data not shown). The constructed graphs contained edges that specified nearest neighbor relations, as based on geometric similarity of Cartesian coordinates of heavy atoms, with edge weights computed by reconstructing each input configuration xi from its k-nearest neighbors and minimizing the reconstruction error εW = Σi (xi−Σj Wijxj)2. The low dimensional manifold that preserved these locally linear neighbor relations was constructed by minimizing εψ = Σi (ψi−Σj Wijψj)2, where ψi is the low dimensional embedding of the high dimensional configuration xi. Such a manifold preserves distance relationships of the data, subject to the constraints of the nearest-neighbor graph and the locally (but not globally) linear embeddings that describe it.
Our approach is related to other graph-based studies of molecular ensembles [61]–[63], but instead of analyzing kinetic or energetic relations among states with respect to each other, we examine their geometric (dis)similarities with respect to the overall organization of the ensemble. In this way, projections of the high dimensional configurations xi, as sampled from their molecular ensembles, onto the low dimensional manifold ψi reveal groups of geometric mesostates and their ensemble coordinates.
We thank Leslie Greengard for helpful discussions, Alex Proekt for comments on the manuscript, and Michael Feig for help with the MMTSB Tool Set. This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov).
Author Contributions
Conceived and designed the experiments: AK RO. Performed the experiments: AK. Analyzed the data: AK TG MM. Contributed reagents/materials/analysis tools: AK. Wrote the paper: AK RO.
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text:
"flandrescarlet1" wrote:
"Ok." said Raichu. Pichu was out of here. "There goes Pichu." said Pikachu.Hi I'm Flandre Scarlet but you can call me Flan-chan
I'm a vampire that's 495 you got a problem?
~For all people who likes/loves touhou and asks me which touhou pairing I like/love it's MariAli~
Neko Marisa Kirisame is cute.
Ask me and Beth!
I'm not always insane...

subset: commoncrawl


text:
BY AIR: El Al, United, Delta, Air Canada and U.S. Airways offer more than 10 daily nonstop flights to Ben Gurion International Airport from New York (JFK), Newark (EWR), Los Angeles (LAX), Atlanta (ATL), Philadelphia (PHL) and Toronto (YYZ). In addition to these carriers, more than 70 international airlines operate flights to and from Israel.
EXCHANGE: Israel's currency is the New Israeli Shekel (NIS), or simply "shekel" in the country's markets, restaurants and attractions. At press time, the exchange rate for $1 was ILS 3.89.
BY WATER: A number of cruise lines make the Israeli ports at Haifa and Ashdod a stop on Mediterranean and Holy Land itineraries, and many of them offer detailed land excursions to the historic cities of Jerusalem and Nazareth. Cruise lines include Princess, Celebrity, Royal Carribean and Oceania.
ON THE GO: Travelers will find ATMs a more ubiquitous and convenient alternative to currency exchange centers. Credit cards are accepted at most shops, restaurants and hotels.
BY LAND: Drivers can enter Israel from Egypt near the southern resort city of Eilat. Travelers may also enter Israel from Jordan via three crossing points along the nation's western border.
NahariyaHwy 6
Lake Tiberias
GROUND TRANSPORTATIONROAD TRIP:Roughly the size of New Jersey, Israelis an easy country to navigate bycar. Road signs in English, Hebrewand Arabic make a cross-country tripa breeze for GPS-armed travelers.Popular rental agencies includingBudget, Avis and Hertz offer rentaloptions for a wide variety of budgets.Taxis are also commonly available inmajor cities.
Hadera Netanya
WHAT'S NEW AT EL-AL AIRLINES It's been a big year for Israel's national airline. Here's a peek at what traveler's can look forward to when they fly El-Al.
Tel Aviv Holon
MORE LEGROOM: El-Al now offers Economy Class Plus seating. With 36 inches of legroom and 33% more recline, designated check-in counters and more, travelers can fly in even greater comfort from New York, London and Paris.
Hwy 6
Jerusalem Jericho
RIDING THE RAILS:Israel's national train network offersaffordable, convenient transportationbetween Tel Aviv, Jerusalem andother popular destinations alongthe Mediterranean Coast and NegevDesert. For more information, visitwww.rail.co.il/EN.
BETTER REWARDS: Economy class fliers can now upgrade to business class at affordable prices by placing a bid on the airline's web site after booking a qualifying flight. El-Al's Matmid frequent flyer program allows travelers to earn bonus flight and upgrade rewards.
Dead Sea
TAKING THE BUS: Egged, the national bus company, operates routes between Tel Aviv and Jerusalem to every city, town and village in Israel, often with dozens of departures daily. Buses operate only sporadically on the Jewish Sabbath (Shabbat). For more information visit www.egged.co.il/eng and www.dan.co.il/english.
Dimona Masada
LUXURIOUS LOUNGES: The King David Club lounges at LAX, JFK and Ben Gurion airports offer complimentary drinks and food, high-speed WiFi service and comfortable seating for business and first class travelers.
CULTURAL CONSIDERATIONS LOCAL TALK: A direct and gregarious people, Israelis don't stand much on ceremony with tourists. That balance of prickliness and warmth is emblematized by their national nickname, "sabra," which refers to the spiny desert cactus whose rough exterior gives way to sweet fruit.
AT YOUR SERVICE Let the Israel Ministry of Tourism (IMOT) help you build an itinerary tailored to your client's needs. Check out www.GoIsrael.com to download regional dossiers and information about major attractions, view sample itineraries and find the representative who serves your area.
LANGUAGE: Most Israelis were born in Israel, but antecedents hail from 100+ countries, which is why everyone speaks Hebrew. Arabic is an official language as well. Travelers will also find everyone fairly fluent in English.
RELIGIOUS OBSERVATION: With the exception of Tel Aviv, many Israeli businesses close from sundown Friday to sundown Saturday in observance of Shabbat, particularly in Jerusalem. Tourists traveling to religious sites are advised to dress modestly.


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Measles is a highly contagious viral disease. It is common to found among children and spreads by sneezing or breathing. The occurrence of this eruptive disease is so common that children all over the globe are found to suffer from the disease. A virus named paramyxo virus causes this disease to occur. It is a very unpleasant and dangerous disease. Measles may lead to Pneumonia in some cases. Measles is declared to be a notifiable disease in the United States. Which means that if any child is found to be suffering from this disease it should be immediately reported to the local government authority or to any health authority. There are a wide variety of home remedies that can be used to treat measles at home without any side effects safely and easily without having to spend much.
1. Invisible droplets of moisture which spreads from the nose or mouth of patient while sneezing or coughing
2. Faulty eating habits
3. Unhygienic condition of living
4. Lower immunity of body
5. Eating food contaminated by flies
1. High fever rising even up to 104 degrees
2. Cold and continuous dry cough
3. Runny nose
4. Sore throat
5. Redness and watering of eyes
6. Swollen lymph nodes
7. Loss of desire to eat or drink
8. Lesser formation of saliva coats on the tongue
9. Diarrhea
10. Delirium
11. Intense toxemia
12. Appearance of eruptions and rashes on the skin after a time period of few days which look like small round spots having reddened skin in between. The initial appearance of it is pink in color and only near the sides of face and neck which gradually turns red in color and spread all over the body.
1. Orange can be very helpful sine it tastes good and thus it helps to overcome the problems like lack of desire to eat or drink for lesser formation of saliva coats or sore throat etc. It is considered to be the best liquid food.
2. Oranges are considered as very valuable home remedies in measles, as it strengthens the seriously hampered digestive power of the body. The intense toxemia and the lack of saliva form a coat on the patient's tongue and destroy his thirst and desire for food. The agreeable flavor of orange juice helps to overcome these bad effects of measles. Orange juice is therefore the ideal liquid food to be taken during measles.
3. Chamomile is a useful herb to cure measles as it can stimulate appetite.
4. The juice of lemon is considered to be the best thirst quenching drink and it overcomes the problem of lack of desire to drink. Take around fifteen to twenty five mili liter of juice of lemon and dilute it with the help of water. This should be drunk continuously in a day leaving a gap of few hours.
5. Turmeric is yet another helpful remedy in the treatment of measles. Take some raw roots of the plant of turmeric and keep them in sun for a couple of days to make them dry. After they are completely dried out grind them to from a fine powder. Take ½ teaspoon of this powder and mix few drops of honey and juice of bitter gourd leaves in it to prepare a mixture. This should be consumed by the patient regularly to cure the disease.
6. If the child suffers with high fever he/she can be made to sit in a tub of lukewarm water and given sponge bath to get relief.
7. To get quick relief from the problem of cough, liquorice is a great help. Take ½ teaspoon of powdered liquorice and mix a few drops of honey to it. This preparation if licked daily by the patient provides great relief in the problem of cough.
8. Barley water is a very fruitful remedy against measles; it should be taken frequently by the patient in a day to treat ma effectively. You can also add newly drawn oil of sweet almonds to sweeten the water.
9. Include garlic and valerian in the diet to be able to fight infection. This is one of the traditional home remedies for measles.
10. Powdered cloves mixed with honey are another useful home remedy in treating measles.
11. To cure burning and itching in measles, use Amla in water, cool and use this water to wash the body.
1. Drink fresh Orange juice had 2 times a day.
2. Eat a light diet. Avoid heavy food and milk products such as cheese. Suggested light foods are Corn flakes and rice flakes.
3. Drink barley water three times a day.


text:
Five Things
{reindeer cupcakes I made for my neighbors}
This week was filled with baking and cleaning. I have a ton of Christmas cookies to decorate this weekend! Next week i'll have some last minute Christmas ideas and something to impress your friends and family with! What are you up to this weekend?
{tacky lawn ornaments}
You have no idea how much I hate lawn ornaments.... these people had at least 100
{wine basket I made for a friend featuring some of my favorite Woozie items}
For the wino in your life be sure to check out the Woozie website. I bought coasters as well as glass and bottle woozie's for gifts this year!
{stuck in a snowglobe}
no snow yet.....but wednesday morning it took over 20 minutes for my vehicle to thaw out
{Rachel Bilson in Hart of Dixie Wearing Of Two Minds Kay Crew Neck Mohair Sweater}
I couldn't find the sweater in purple, but I did find it on sale in orange.
I did find the cardigan in purple and it is on sale!
24 comments :
Oh my gosh, how cute are those reindeer cupcakes! So creative. LOL. I think I'm going to make some too for my Christmas party with my friends. Thanks for the idea! Like you, I'm alsot not a fan of lawn ornaments or decorations. I just like putting out the traditional Christmas lights and maybe one of those reindeers that lights up. I think when I get my own house, I'm going to fill it up with lots and lots and lots of Christmas lights.
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Alevi Aprons are an Idakoos specialtiy! Idakoos has many things to offer. Whether you want to get a Alevi Apron for a special occasion, give a Alevi Canvas Apron as a present or need to represent with a funny Alevi Apron during Holidays or Family and Friends get-togethers, Idakoos is sure to have the right Alevi Apron for you! Even if it turns out you can't find the right Alevi Canvas Apron with the perfect design for your mother, father or children (or friends!); Idakoos offers all of its Alevi Aprons as Custom Alevi Aprons. Personalize any Alevi Apron the way you want to ensure the perfect gift.
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First attempt at levitation. Meh.
2 responses
christopher kontoes (Deleted) said (6 Jun 2010):
Paul Lavallee gave props (17 Jun 2010):
wild! (no pun intended)
Also by Chelsea Wilde


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Selecting your vehicle's model and year allows us to display only parts and items specific to your car's model and year.
Part# CH25296
Mfg# 32260
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OPGI Price: $99.99
Brand: MILODON Part Type: (P) High Performance, designed to improve performance BB = Big-block engine Years: 1978-88 Sold As: EACH 2017 Late El Camino Catalog Page: 260
Everyone wants more power. One of the easiest ways to get it is to install the Milodon "Diamond Stripper" windage tray. Any windage tray is good, but with a factory, or solid-style tray, you get what's called "bounce back", as the slinging oil hits the solid tray and bounces back up into the rotating crank and rods, causing a loss of power. With the Diamond Stripper, hundreds of tiny holes in the tray trap the oil and drop it down into the oil pan, with no "bounce back" and a gain in power. The results have been proven on the dyno and race track many times. On small-block engines the Diamond Stripper will fit left hand and right hand dipstick applications when the tray is adjusted properly.


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The Lady Plumber
Fiction, Consensual Sex, Female/Female, Male / Females, Threesome
Author's infos
Gender: male Age: N/A Location: N/A
Posted Sat 12th of May 2012
Our plumber wears makeup and short skirts.
Fbailey story number 734
The Lady Plumber
My wife had been complaining about a drip in the kitchen faucet. Of course I kept ignoring her.
So one day I came home from work, walked into the kitchen, and saw what I thought was my wife lying on her back under the sink.
She had on a pair of black high heels, a tan colored miniskirt, and a bright yellow T-shirt that showed off her belly button piercing. When did she get her belly button pierced? Why was she dressed to impress? Why was she under the sink? She didn't know what she was doing.
Her knees were up and they were spread, exposing her pretty pink and blue panties. Her camel toe was showing. I knelt down and placed my hand on her warm pussy. She jumped and hit her head on a pipe under there.
Then from behind me my wife asked, "Why are you feeling up our new lady plumber?"
From under the sink a voice said, "Don't make him stop, I was just beginning to enjoy it."
What to hell…I placed my hand back on her panty covered pussy and said, "Most plumbers show their ass crack…your camel toe is much better."
My wife said, "Maggie, he likes your slit."
Maggie said, "So did my husband until he saw how cute his boyfriend's butt crack was. After that my slit didn't excite him anymore."
I slipped a finger under the side of her panties and then I slipped it up her crack from her asshole to her clit. I stayed on her clit and rubbed my index finger into her until she lowered her arms over her head and started to moan.
My wife was standing right over me looking down as she said, "Maggie, I'll be expecting a discount."
Maggie said, "Then I'll be expecting more than just him fingering me."
My wife started laughing and said, "Okay, I'll make a deal with you. You take care of my drippy sink and I'll take care of your drippy pussy after my husband fills you up…lets say…three times tonight."
Again Maggie hit her head when she jumped, her arms went to her forehead, and she said, "Three times…girl…are you serious?"
My wife replied, "Damn serious. My husband is the best lover that I've ever had. Of course I've only had eleven but he was and is, the last."
Maggie giggled and asked; "Don't I count?"
My wife squatted down and reached between my legs. Soon her fingers were in Maggie's pussy hole and she was saying, "No, women don't count. You can't find me, feel me up, fuck me, and then forget me…like most men do."
Maggie finished her job once I stopped distracting her. When she came out and stood up I was amazed at how pretty that woman was.
I said, "You are a very attractive plumber. Do you always dress like that?"
Maggie had a great smile as she replied, "I was on my way to meet my sister for dinner when your wife called me. I thought I could do this on my way…but now…I think I'll call my sister and tell her that something better came up." She said the last part as she cupped my crotch.
The phone call was made and I followed the two women into our spacious bedroom. It was certainly no ordinary bedroom. Our king size bed was in the middle and could spin around on casters…slowly of course…I wouldn't want anyone to get sick or get thrown off. There was a Jacuzzi in one corner and next to it was six feet of our forty-foot swimming pool. It was heated and we could swim all year round. Then along another wall was an open bathroom. It had a sunken tiled floor to contain any water from the double sinks, toilet, bathtub, and large shower. Everything was fully visible to everyone else in the bedroom. Even I would pee from the side so as not to encroach upon on my wife's view of my cock as I peed.
Maggie loved our setup. I loved watching her undress with my wife. It was blouse for blouse, bra for bra, and skirt for skirt. They let me remove their damp panties.
My wife kissed Maggie and got her just as hot as she had been in the kitchen. I thrust my cock into her willing hole and pumped her softly, apparently too softly, because she begged me to fuck her harder. I had been excited too long to stretch it out much longer and when I erupted inside her it was powerful.
My wife jumped in and licked her clean quickly. Then we took a swim, sat in the Jacuzzi, and took a shower together. As I watched the two women soap each other up and then finger the other's pussy, I got hard. I lifted Maggie up against the wall, positioned my hard cock under her pussy, and then I lowered her down onto it. With her arms and legs wrapped around me I slammed her into the wall and pounded her with my hips. Her ass was forced into the wall repeatedly until she had her orgasm…followed by my climax. Again my wife was right in there licking and washing her bald pussy.
The girls let me take a nap. Maggie woke me up by placing her juicy pussy over my mouth. What a fantastic way to wake up. Anyway my wife had promised Maggie three good size deposits in her pussy and Maggie did not want to get cheated. She didn't.
We never had another problem with our plumbing. Maggie became a regular visitor to our house, to our bedroom, and of course to our bed. She brought her sister along several times. I loved watching the three beautiful women enjoying each other sexually.
The End
The Lady Plumber
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The talks between the P5+1 and Iran got off to a promising start. Not surprisingly, that doesn't please Benjamin Netanyahu. I explore at LobeLog.
Posts Tagged 'Yom Kippur War'
Delegitimizing Israeli Democracy
Posted in Democracy, human rights, tagged al-haq, B'Tselem, Bashar al-Assad, Benjamin Netanyahu, East Jerusalem, Flytilla, Gaza Strip, Gisha, Hamas, Iran, Israel, Palestine, Six Day War, Syria, West Bank, Yom Kippur War, Zionism on April 18, 2012| Leave a Comment »
In this week's entry at Souciant, I examine the implications of Israel's heavy-handed, stupid and clumsy response to the intention of hundreds of activists to fly into Israel in order to join a Palestinian protest. The ironic thing is that Netanyahu trots out the standard "Israel is the region's only democracy" argument to defend actions that both show how deeply flawed that democracy is and how seriously that democracy is threatened.


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A disciple of Dracula. Oh my God!
Professor Van Helsing
Do old ideas rest comfortably in an open mind?
Professor Van Helsing
Jessica, don't go near him! For God's sake, don't touch him!
Professor Van Helsing
My grandfather died fighting a vampire. He was the most terrible, the most dangerous vampire of all time.
Professor Van Helsing
You would play your brains against mine? Against me who has commanded nations?
Count Dracula
I have returned to destroy the house of Van Helsing forever, the old through the young. You and your line have been chosen.
Count Dracula
Dig the music, kids. Let it flow into you.
Johnny Alucard
Look, just to show I'm sorry, I got two tickets for the Jazz spectacular at the Albert Hall.
Johnny Alucard
Okay, okay. But if we do get to summon up the big daddy with the horns and the tail, he gets to bring his own liquor, his own bird, and his own pot.
Joe Mitcham
Okay, okay, so we'll go to the cavern tomorrow, usual time. Laura will be there. A bit drained, but she'll be there. And I'll have a few words to say to Johnny.
Bob Tepper


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The Anthropic Cosmological Principle
It has been observed that cosmologists are often wrong but seldom uncertain, and the authors of this long, fascinating, exasperating book are no exceptions. They are John Barrow, astronomer at the University of Sussex, and Frank Tipler, Tulane University mathematical physicist. Physicist John Wheeler provides an enthusiastic foreword. No one can plow through this well-written, painstakingly researched tome without absorbing vast chunks of information about QM (quantum mechanics), the latest cosmic models, and the history of philosophical views that bear on the book's main arguments.
Just what is this "anthropic principle" that has become so fashionable among a minority of cosmologists, and is arousing such passionate controversy? As the authors make clear in their introduction, there is not one principle but four. Each is more speculative than the previous one, with the fourth blasting the authors out of science altogether into clouds of metaphysics and fantasy.
The simplest of the four is called (the authors are fond of acronyms) WAP, or the Weak Anthropic Principle. Although it goes back to Protagoras's famous declaration that "man is the measure of all things," its modern cosmological form seems first to have been stated by the physicist Robert Dicke in the late 1950s. As Barrow and Tipler readily admit, it is a trivial tautology, totally noncontroversial. It merely proclaims that because we exist the universe must be so constructed as to allow us to have evolved. The laws of nature clearly must be such as to permit, if not actually force, the formation of CHON (carbon, hydrogen, oxygen, and nitrogen), the four elements essential to life as we know it.
Does this mean that all life must be carbon based? Although the authors believe this, it does not follow from WAP. Even if there is noncarbon life elsewhere in the universe, the fact that we are carbon imposes a variety of tight restraints on the universe and its past. For example, the cosmos has to be about 15 billion years old. Why? Because, the authors argue, elements necessary to organic molecules are cooked inside stars. If the universe were much younger, those elements would not be available and we wouldn't be here. If the universe were much older, all the suns would have burned out, and we wouldn't be here either.
WAP was invoked over and over again in earlier centuries by proponents of the design argument for God. It was WAPish to point out that if the earth were slightly closer to the sun, like Venus, water would boil away and carbon life would be impossible. If the earth were slightly farther from the sun, water would freeze and Earth would have the barren deserts of Mars. Theists liked to note that when water freezes it expands and floats on water, otherwise lakes and rivers would freeze to the bottom in winter and all their life be destroyed. If Earth did not have an ozone atmosphere, animals could not survive ultraviolet radiation. And so on. Hundreds of similar arguments, most of…
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HitRoach HD
- App Store Info
HitRoach is a funny game. If you really hate cockroache and want to kill them, I think you will like to play the HitRoach game. Have fun!What's New in Version 1.2
1. Improved the performance of HitRoach game.2. Added a new level 6.
3. Adjusted levels:
level1: 200
level2: 500
level3: 800
level4: 1200
level5: 1600
level6: no limit


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Join my instagram obsession
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Look cute, do good! Proceeds to charity.
Daily Archives: May 5, 2011
Is It Thursday … Or "Sun" Day
Look look, the sun is trying to make it into my backyard. Try hard sun, try hard! I've got flowers to keep alive! Did you notice that deja vu breakfast? More oranges, they're just so good. Also, Brad and I … Continue reading
Posted in Gardening, Healthy Eating, Student Life
Tagged flower garden, May agenda, Women Biz Network
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During the past couple of years, and since before the war, Hollywood movies have been getting worse and worse. Very few pictures are receiving the unequivocal praise which, at the beginning of the thirties, used to flow regularly from the pens of the critics, and box office receipts have fallen off decidedly. There can be no doubt about it. Hollywood is in a bad way.
Different people have different hates about the movies. Some say the great old actors are vanishing from the scene, and in their stead are appearing a group of highly publicized, incompetent "starlets"; the John Barrymores are giving way to the Ann Sheridans. Home blame the script writers, some the directors. But though each of these arguments may be perfectly valid in regard to specific faults of the movies, they do not arrive at the basic cause for Hollywood's declining standards. This fundamental cause is the belief shared by practically everyone in Holywood, that the movie-going public has an I.Q. a little lower than that of a mentally retarded twelve-year-old.
Of course, not all pictures are made on this theory. But the success of those few intelligent movies which Hollywood produces only strengthens the case of the advocates of a more advanced I.Q. for the persecuted moviegoing public, and perhaps some day it will persuade Hollywood that movies can be an uplifting rather than a degrading force in American life. There are innumerable fields of cultural entertainment as yet untapped by the movie moguls. All the great literature from Homer to Galsworthy lies open; the copyrights are for the most part non-existent; the entertainment value is unquestioned. The only debatable factor is the American intelligence; and America, by its cold reception of a great many of the flowers of the latest crop of Holywood output, has pretty well proved that it is past the stage of adolescence.
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Japan Society Presidents Elected


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The Horse Forum
- Horse Health
- - Giving a horse a new home
Giving a horse a new home I know someone who is having financial difficulty and wants to give their quarter horse a new home, a safe secure home. I am from the Chicago land area and was wondering if anyone had any ideas. |
Try Rescue Organizations. They take horses in from situations quite like theirs. If you'd like I can search for some in your area. |
If you dont mind I would really appreciate it. I tried to find some but i had no luck. thanks alot!!! |
http://www.crosswindseqresq.org/ http://www.harpsonline.org/ http://www.hahs.org/ http://www.saplonline.org/horses_ahspasupp.htm These are all in Illinois. The last one has a LOT of info to look through. Good luck and keep us posted. [/url] |
You can ask around at some nicer boarding stables to see if one of their clients wants to lease the horse. |
Depending on the severity of their problems they could also sell them or give them to someone, particularly if it's a long term issue. If there's nothing wrong with the horse I'd try selling it first. Give it a try for a couple of weeks. If nothing turns up then I'd turn them over to a rescue. My rationalizing is that rescue organizations need to spend the most time and money on the horses they rescue from such places as abusive homes. If they can avoid putting extra strain on the rescues that would be best. I'd take the horse if I could. :) |
There is a great rescue in Sandwich, IL (about 60 miles from Chicago). I don't remember what it is called but I think it is something like Lazy Maple. The one thing about a rescue is that they will not take a horse if it is not trained to ride--they are just so much easier to place. If your friend has time now is definitely a good time to try to sell the horse. There are a lot of online horse ad's. IL horse online would be a good place for someone in the Chicago area. Good Luck |
Thankyou to everyone who replied. We actually gave her away for free to a very nice lady who has other horses and an 80 acre farm! Shes an awesome person and keeps in touch via E-mail and has given us the okay to visit! Thanks again! |
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New KCBS Scoring Program Test
View Single Post
Got Wood.
Join Date: 12-16-10
Location: Phoenix,az
Originally Posted by
That might be an interesting idea. Talking to reps, they sometimes KNOW there are certain judges that always score low or score high. Some reps have told me they dread certain judges showing up at a contest because they know a lot of 5s and 6s are going to be handed out for some good bbq. The reps, however, don't have any power to DO anything about that. The above suggestion might have to mitigate some of the risk - but I wouldn't try to group like judges together.
Winning the table...when it's a low scoring table...doesn't help you. Spreading out low scoring judges evenly across all the tables would be more fair to the teams.
This brings up some food for thought. Why is it that someone scoring 5-6 is considered "low scoring"? Doesn't the rating scale go from 2-9 or has this truly turned into 6-9 scoring? Is there now an attitude like in T-Ball where there's a trophy for just being there?
The reps said "they know a lot of 5-6's are going to be handed out for some good BBQ", in whose opinion (theirs?) and why is their opinion any more valid that the person judging it?
Then you have the question of who's palate is correct, could the people who're "low scoring" actually have a better palate than the "high scoring"?
Would y'all prefer false high scoring? If so then maybe the scoring and judges instructions should be changed to reflect that.
I hope you don't take this as me jumping on you or what you posted, it's not meant that way, I think you bring up some good points for discussion.
I keep hearing how this judge or that judge scored low so the judge "must" be the issue, why isn't the same said for judges that score crazy high when the rest of the table doesn't? I've never,ever seen a judge taken aside for scoring too high, just too low.
A good example of this whole argument is Hells Kitchen. You have all the chefs make a dish and invariably each one thinks it tastes fantastic. When it's tasted by Gordon or a selected group of celebrity chefs they have a way different opinion. Same thing on Chopped,Cupcake wars,wine tasting etc.
Until such a time where an absolute set of iron clad taste criteria can be laid out so every person tastes the same way/thing AND the judges are all instructed the absolute same way then you're going to have personal taste variations/rule interpretations that are not explainable nor changeable.
I guess one thing could be done which is having to pass a palate test before you're allowed to be a judge.
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Defense Building Against ObjectVideo's USITC Case
Author: John Honovich, Published on Oct 02, 2011Login to read this IPVM report.
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Tue 17 Apr 2012
Prodigy Speaks on Mobb Deep Rumors with Cipha Sounds & Rosenberg (Video)
Posted by Mr.North under Interviews[6] Comments
Prodigy paid a visit to Hot 97's Cipha Sounds and Rosenberg Morning Show recently to speak on all the rumors surrounding Mobb Deep. As Havoc did an interview last week, Prodigy says the rumors of the two breaking up isn't true. He also mentions tweets made on Havoc's account stating certain allegations are also false, and speaks on the rumors of him possibly dating Rihanna. The full interview airs tomorrow morning.
Brotherly Love: Mobb Deep Attend NY Knicks Game Together (Photo)
Mobb Deep Dischord: Havoc's and Prodigy's Twitter Statements (UPDATE: Havoc Releases Statement)
6 Responses to "Prodigy Speaks on Mobb Deep Rumors with Cipha Sounds & Rosenberg (Video)"
slice_w/o_cheese Says:
April 17th, 2012 at 10:28 pm"WE DONT BELIEVE YOU…YOU NEED MORE PEOPLE"
RoeLuv Says:
April 18th, 2012 at 4:37 amco-sign
yoprince Says:
April 18th, 2012 at 8:22 amit was a stunt. and it's lame.
they tried throwing everything at the wall:
1. hookup with celebrity (Rihanna)
2. Beef (the two of them fighting)
3. Gay RumorsI don't think I can rock with these dudes after this.
jwilliams Says:
April 18th, 2012 at 12:18 pmi don't think prodigy signed up for gay rumors. Lol. i think havik was jealous and upset about the book side project (side money), and decided to smear his partner. prodigy probably went to the idiot and set things straight. envy.
ChicagoMadeMe Says:
April 18th, 2012 at 1:49 pmThis is corny.. Prodigy sounds so uncomfortable in this interview. A few years back, you couldn't tell me they would partake in this bullshit in the future. Sad
ChicagoMadeMe Says:
April 18th, 2012 at 5:01 pmHavoc get "side money" too.. he's a producer.


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Wednesday, April 20, 2011
Fraudulent Tax Returns Surge 181% This Year
CNN, Fraudulent Tax Returns Surge 181%:
The number of taxpayers trying to bamboozle the IRS and collect bigger refunds has shot up this filing season. The IRS identified 335,341 tax returns claiming $1.9 billion in fraudulent refunds as of March 4, 2011, according to the findings of an audit conducted by the Treasury Inspector General for Tax Administration. That's a whopping 181% increase from the same period last year.


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No Mugshot Available
Who is Masoud Shojaee? Was Masoud Shojaee arrested? When was Masoud Shojaee arrested? What was Masoud Shojaee arrested for? Is this a mugshot of Masoud Shojaee? Are there more Mugshots of Masoud Shojaee? Are Mugshot laws constitutional? Who decides which Mugshots are Indexed? Why must Mugshots remain public mugshots and public record? Is this the Masoud Shojaee you know?
Masoud Shojaee was booked in Miami-Dade County, FL.
All are presumed innocent until proven guilty in a court of law. Published mugshots and/or arrest records are previously published public records of: an arrest, an indictment, a registration, the deprivation of liberty or a detention. The mugshots and/or arrest records published on Mugshots.com are in NO way an indication of guilt and they are NOT evidence that an actual crime has been committed. For latest case status, contact the official law enforcement agency which originally released the information.
The following Official Record of Masoud Shojaee is being redistributed by Mugshots.com and is protected by constitutional, publishing, and other legal rights. This Official Record was collected from a Law Enforcement agency on 12/17/2012.
Mugshots.com ID: 32279170
Court Case No: F82014921
State Case No: 131982CF0149210001XX
Name: SHOJAEE, MASOUD
Date of Birth: 8/03/1959
Date Filed: 6/16/1982
Date Closed: 7/06/1982
Warrant Type: N/A
Assessment Amount: $0.00
Balance Due: $0.00
Stay Due Date: N/A
Hearing Date: N/A
Hearing Type: N/A
Court Room: REGJB - JUSTICE BUILDING, ROOM No.: 7-4
Court Address: 1351 N.W. 12 ST
Previous Case: N/A
Next Case: N/A
Judge: FERNANDEZ JOSE L
Defense Attorney: N/A
Bfile Section: F003
File Location: DESTROYED
Box Number: N/A
Probation Start Date: N/A
Probation End Date: N/A
Probation Length: N/A
Probation Type: N/A
Defendant in Jail: Y
Defendant Release To: N/A
Bond Amount: N/A
Bond Status: N/A
Bond Type: N/A
Bond Issue Date: N/A
Dockets: N/A
External Links:
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NILAM (Nadi Ilmu Amalan Membaca) is the official name as a program organized by Educational Technology Division (ETD). This program running at the primary and secondary government schools with is supervision by the Teacher Training Center (TTC) is one of the networks under ETD. Under this program, ETD should takes responsibility NILAM's program able to carry out by the schools with smoothly. Regarding that, it should provide training and service to the teachers and collecting the report and data related with NILAM and do some analysis work before submit it to upper management.
A strategy to grow interest in reading by organized and arranged among Malaysians, especially schoolchildren until to having reading habit and culture reading among Malaysians.
Make pupil read more including all field, quality material, various genre instead of readings material oriented examination only.
Encourage school generate continuously creative and innovative idea to inculcate this reading habit.
The outline of this program is simple enough, where each pupil is asked to:
1. Record the books they had read in their Record Book NILAM (can be found in any public school's book store)
2. Get their teachers to check their records
3. Whoever reads the most books gets a reward
"Pupil successfully completed NILAM's program have privileged for elections to Limited Schools, Fully Residential Schools, Express Class, scholarships sponsoring and Higher Learning Institution."
- National Library of Malaysia
Implementation level and NILAM achievement
NILAM's implementation program divided into two-stage:
- Jauhari's Level:
Read many book and recorded. Recorded based on number, subject, and writer and also write a little summary.
- Reader Partner:
Eligible implement readings activity after reading 100 book and above.
Number of books to be read and documented in order to receive the NILAM award
Number of books read | Honors |
90-179 | Bronze |
180-269 | Silver |
270-359 | Gold |
360 and above | NILAM |
Number of books read | Honors |
72-143 | Bronze |
144-215 | Silver |
216-287 | Gold |
288 and above | NILAM |
The NILAM program is supposed to be compulsory for every school children but most schools does not take this activity very seriously because they would rather the children read academic books instead. Therefore, we at Q'reazant feel that it is up to us to encourage Malaysian children to go for this award; hence the formation of Q'reazant Reading Class.


text:
Speedo Aquapulse Max 2The Speedo Aquapulse Max goggles feature Speedos revolutionary IQ Fit Technology, which is engineered to fit real face contours, and include a unique 3D seal which offers maximum leak resistance even during starts and turns.Aquapulse Max Goggles have been designed for those who take swimming seriously, for training or for keeping fit. Wide lenses offer maximum vision whilst the Anti-fog coating ensures clear vision and clarity underwater. The result is a precision fit which gives you the freedom to achieve your best in the water.Features: Material: 100% Polyester Lens: Polycarbonate Seal: Thermoplastic rubber Wishbone: Thermoplastic polyurethane Strap: Silicone Part of the Fitness Training range Ideal for fitness swimming or training and regular swimming Speedos first fitness goggle to utilise the IQ Fit technology originally developed for their top racing goggles IQ Fit uses body scanning research, so Speedo are able to engineer products designed for
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