Position of helical kinks in membrane protein crystal structures and the accuracy of computational prediction

The structural features of helical transmembrane (TM) proteins, such as helical kinks, tilts, and rotational orientations are important in modulation of their function and these structural features give rise to functional diversity in membrane proteins with similar topology. In particular, the helical kinks caused by breaking of the backbone hydrogen bonds lead to hinge bending flexibility in these helices. Therefore it is important to understand the nature of the helical kinks and to be able to reproduce these kinks in structural models of membrane proteins. We have analyzed the position and extent of helical kinks in the transmembrane helices of all the crystal structures of membrane proteins taken from the MPtopo database, which are about 405 individual helices of length between 19 and 35 residues. 44% of the crystal structures of TM helices showed a significant helical kink, and 35% of these kinks are caused by prolines. Many of the non-proline helical kinks are caused by other residues like Ser and Gly that are located at the center of helical kinks. The side chain of Ser makes a hydrogen bond with the main chain carbonyl of the i − 4th or i + 4th residue thus making a kink. We have also studied how well molecular dynamics (MD) simulations on isolated helices can reproduce the position of the helical kinks in TM helices. Such a method is useful for structure prediction of membrane proteins. We performed MD simulations, starting from a canonical helix for the 405 TM helices. 1 ns of MD simulation results show that we can reproduce about 79% of the proline kinks, only 59% of the vestigial proline kinks and 18% of the non-proline helical kinks. We found that similar results can be obtained from choosing the lowest potential energy structure from the MD simulation. 4–14% more of the vestigial prolines were reproduced by replacing them with prolines before performing MD simulations, and changing the amino acid back to proline after the MD simulations. From these results we conclude that the position of the helical kinks is dependent on the TM sequence. However the extent of helical kinking may depend on the packing of the rest of the protein and the lipid bilayer.     

An expert protein loop refinement protocol by molecular dynamics simulations with restraints

Protein structure prediction (PSP) is a long standing problem in structural biology and bioinformatics. Within the PSP problem loop refinement is a major bottleneck. In this article we report the latest version of the CReF expert predictor system for the PSP problem with emphasis on loop refinement of the approximate 3-D structure 1ZDD_P of the Z34C mini protein predicted by CReF. We designed a loop refinement protocol based on seven molecular dynamics (MD) simulations runs at different temperatures. We found that, by letting the loop residues move freely during dynamics at 325 K and restraining the internal coordinates of the correctly predicted helical structures, while allowing them to move relative to each other, the refinement protocol was very effective in predicting an accurate loop conformation in the first 100 ps of a 1000 ps MD simulation. The quality of the predictions was confirmed by the RMSD between refined and experimental structures which varied from 0.6 to 1.3 Å. In addition, stereochemical analyses showed that 100% of all residues of the refined 1ZDD_P, including those in the loop, populates the most favorable core regions of the Ramachandran plot. Our study suggests that the proposed protocol may be suitable to refine more complex mini proteins with different classes and architectures.     

Investigating detailed interactions between novel PAR1 antagonist F16357 and the receptor using docking and molecular dynamic simulations

Currently, Vorapaxar is the only recently FDA-approved antiplatelet drug targeting Protease-activated receptor 1 (PAR1). However, a novel antagonist, F16357, has been shown to prevent painful bladder syndrome, also known as interstitial cystitis (IC). Unfortunately, there is no high resolution structure of the F16357-receptor complex, hindering its optimization as a therapeutic agent. In this study, we used docking and molecular dynamic (MD) simulations to investigate the detailed interactions between F16357 and PAR1 at a molecular level. The recently solved crystal structure of human PAR1 complexed with Vorapaxar was used in our docking of F16357 into the binding pocket of the receptor. To enhance binding pose selection, F16357 was docked first without constraints and then with a positional constraint to invert its orientation to become similar to that of Vorapaxar. The three systems, with crystal Vorapaxar, F16357 and an inverted F16357, were subjected to 3.0 μs MD simulations. The MM-GBSA binding energy analysis showed that F16357 binds more strongly in a pose obtained from an unrestrained docking than in the inverted pose from a restrained docking; and Vorapaxar binds more strongly than F17357. This ordering is consistent with the experimental pIC₅₀ values. Our structural data showed subtle changes in the binding pose between Vorapaxar and F16357. Transmembrane helices 1, 2, 5, and 7 were most significantly affected; most notably a large kink at F279^5.47 in TM helix 5 of the Vorapaxar complex was completely absent in the F16357 complex. The results of this study facilitate the future development of other therapeutic PAR1 antagonists.     

On the mechanism of substrate/non-substrate recognition by P-glycoprotein

P-Glycoprotein (P-gp, multi-drug resistance protein, MDR1) plays a gatekeeper role, interfering delivery of multiple pharmaceuticals to the target tissues and cells. We performed Molecular Dynamics (MD) simulations to generate fifty side-chain variants for P-gp (PDB ID: 4Q9H-L) followed by docking of 31 drugs (0.6 ≤ ER ≤ 22.7) to the whole surface except the ATPase domains and the extracellular part. A selection of the most negative energy complex for each ligand followed. All compounds docked to the two areas – the main binding cavity at the top of P-gp (12.5% of compounds with ER < 1; 44.4% of 1 ≤ ER ≤ 2; and 100% of ER > 2), and the binding sites in the middle of P-gp (87.5% of ER < 1; 55.6% of 1 ≤ ER ≤ 2; and 0% of ER > 2). Our results show that anti-substrates (ER < 1), intermediate compounds (1 ≤ ER ≤ 2) and strong substrates (ER > 2) might behave differently in relation to the P-gp. According to our calculations, the anti-substrates almost do not bind the main binding cavity (MBC) of P-gp and rather approach the other binding sites on the protein; the substrates preferably bind the MBC; the intermediate compounds with 1 ≤ ER ≤ 2 bind both MBC and other binding sites almost equally. The modelling results are in line with the known hypothesis that binding the MBC is prerequisite for the pumping the compound off the P-gp.     

Separation based adsorption of ethanol–water mixture in azeotropic solution by single–walled carbon,boron-nitride and silicon-carbide nanotubes

The separation of the azeotropic ethanol-water mixture (95.57 wt% ethanol) over a wide range of pressures (100–100000 kPa) was studied on armchair SWCNTs, SWSiCNTs and SWBNNTs with different diameters at 351.30 K using GCMC simulations. The GCMC results demonstrated that ethanol and water molecules form a monolayer single-file, chain together in the center of (6,6) SWCNT, while a spiral ring of ethanol and water is formed in the center of (8,8), (10,10) and (12,12) SWCNTs. It was found that in SWCNTs, the adsorption of ethanol reduces the function of pressure, while water adsorption increases its function. Water selectivity rises as a function of pressure. Also, in SWBNNTs, the adsorption of water increases as a function of pressure, while ethanol adsorption is almost constant. However, in the case of SWSiCNTs, ethanol and water adsorptions are very similar to those of SWBNNTs, whereas the adsorptivities of SWSiCNTs are more than those of SWBNNTs. Our findings regarding adsorption and slope of adsorption indicate that higher pressures are favorable for separating water and ethanol by SWCNTs, while SWBNNTs and SWSiCNTs are demonstrate higher ethanol adsorptivities in lower pressures. Also, MD simulations have been performed to study the microscopic structure and diffusion of binary mixtures of water and ethanol within SWCNTs, SWSiCNTs and SWBNNTs. The MD simulations imply that the oxygen atoms are highly well-organized around themselves. Also, the MD results illustrate a similar tendency for oxygen of water (OW) and oxygen of ethanol (OE) to the wall of the nanotubes in all the pressures. In addition, from the MD results, self-diffusion of water and ethanol in all nanotubes were calculated and discussed.     

Deconstruction of the human connexin 26 hemichannel due to an applied electric field; A molecular dynamics simulation study

Connexins are a 21-member membrane protein family constituting channels evolved in direct communication between adjacent cells by passaging cytoplasmic molecules and ions. Hexametrical assembly of connexin proteins in plasma membrane forms a wide aqueous pore known as connexin hemichannel. These hemichannels mediate cytoplasm and extracellular milieu communication both in many external tissues and in the central nervous system. In this study, a series of molecular dynamics simulations has been performed to investigate the effect of applied static and alternating electric fields on the stability and conformation of human connexin26 hemichannel. The root mean square deviations of C-alpha atoms, the dipole moment distribution, the number of inter-protein hydrogen bonds and the number of water-protein hydrogen bonds were used to assess connexin26 hemichannel stability. In the static field case, our results show that although the lowest field used in this study (0.1 V/nm) does not lead to the hemichannel deconstruction, stronger fields (>0.1 V/nm), however, disrupt the protein structure during the simulations time period. Furthermore, in the alternating case, compared to static field case, field effects on the connexin26 hemichannel conformation are reduced and consequently the protein maintains its native structure for longer times. Specifically, for the highest frequency used in this study (50 GHz), the hemichannel keeps its structure even under the effect of the strongest field (0.4 V/nm). According to our results, the protein secondary structure is preserved in the characteristic times determined for the protein deconstruction. Consequently, we suggest that the protein deconstruction is due to the tertiary and quaternary structure loss.     

Solvent effect on the crystal morphology of 2,6-diamino-3,5-dinitropyridine-1-oxide: A molecular dynamics simulation study

The attachment energy (AE) calculations were performed to predict the growth morphology of 2,6-diamino-3,5-dinitropyridine-1-oxide (ANPyO) in vacuum. The molecular dynamics (MD) method was applied to simulate the interaction of trifluoroacetic acid solvent with the habit faces and the corrected AE model was adopted to predict the growth habit of ANPyO in the solvent. The results indicate that the growth morphology of ANPyO in vacuum is dominated by (1 1 0), (1 0 0), (1 0 −1) and (1 1 −2) faces. The corrected AE energies change in the order of (1 1 0) > (1 0 −1) > (1 1 −2) > (1 0 0), which causes the crystal morphology to become very close to a flake in trifluoroacetic acid solvent and accords well with the results obtained from experiments. The radial distribution function analysis shows that the solvent molecules adsorb on the ANPyO faces mainly via the solvent–crystal face interactions of hydrogen bonds, Coulomb and Van der Waals forces. In addition to the above results, the analysis of diffusion coefficient of trifluoroacetic acid molecules on the crystal growth faces shows that the growth habit is also affected by the diffusion capacity of trifluoroacetic acid molecules. These suggestions may be useful for the formulation design of ANPyO.     

Characterization of CO2 and mixed methane/CO2 hydrates intercalated in smectites by means of atomistic calculations

The recent increase in anthropogenic CO₂ gas released to the atmosphere and its contribution to global warming make necessary to investigate new ways of CO₂ storage. Injecting CO₂ into subsurface CH₄ hydrate reservoirs would displace some of the CH₄ in the hydrate crystal lattice, converting simple CH₄ hydrates into either simple CO₂ hydrates or mixed CH₄CO₂ hydrates. Molecular simulations were performed to determine the structure and behavior of CO₂ and mixed hydrate complexes in the interlayer of Na-rich montmorillonite and beidellite smectite. Molecular Dynamics (MD) simulations used NPT ensembles in a 4 × 4 × 1 supercell comprised of montmorillonite or beidellite with CO₂ or mixed CH₄/CO₂ hydrate complexes in the interlayer. The smectite 2:1 layer surface helps provide a stabilizing influence on the formation of gas hydrate complexes. The type of smectite affects the stability of the smectite-hydrate complexes, where high charge located on the tetrahedral layer of the smectites disfavor the formation of hydrate complexes.     

Passive single chip wireless microwave pressure sensor

A novel micromachined passive wireless pressure sensor is presented. The device consists of a tuned circuit operating at 10 GHz fabricated on to a SiO₂ membrane, supported on a silicon wafer. A pressure difference across the membrane causes it to deflect so that an antenna circuit detunes. The circuit is remotely interrogated to read off the sensor data wirelessly. The chip area is 5 mm × 4 mm and the membrane area is 2 mm² with a thickness of 4 μm. Two on-chip passive resonant circuits were investigated: a meandered dipole and a zigzag antenna. Both have a physical length of 4.25 mm. The sensors show a shift in their resonant frequency in response to changing pressure of 10.28–10.27 GHz for the meandered dipole, and 9.61–9.58 GHz for the zigzag antenna. The sensitivities of the meandered dipole and zigzag sensors are 12.5 kHz/mbar and 16 kHz/mbar respectively.     

Polyelectrolyte conformational transition in aqueous solvent mixture influenced by hydrophobic interactions and hydrogen bonding effects: PAA–water–ethanol

Molecular dynamics simulations of poly(acrylic acid) PAA chain in water–ethanol mixture were performed for un-ionized and ionized cases at different degree-of-ionization 0%, 80% and 100% of PAA chain by Na⁺ counter-ions and co-solvent (ethanol) concentration in the range 0–90 vol% ethanol. Aspects of structure and dynamics were investigated via atom pair correlation functions, number and relaxation of hydrogen bonds, nearest-neighbor coordination numbers, and dihedral angle distribution function for back-bone and side-groups of the chain. With increase in ethanol concentration, chain swelling is observed for un-ionized chain (f = 0) and on the contrary chain shrinkage is observed for partially and fully ionized cases (i.e., f = 0.8 and 1). For un-ionized PAA, with increase in ethanol fraction ϕ_eth the number of PAA–ethanol hydrogen bonds increases while PAA–water decreases. Increase in ϕ_eth leads to PAA chain expansion for un-ionized case and chain shrinkage for ionized case, in agreement with experimental observations on this system. For ionized-PAA case, chain shrinkage is found to be influenced by intermolecular hydrogen bonding with water as well as ethanol. The localization of ethanol molecules near the un-ionized PAA backbone at higher levels of ethanol is facilitated by a displacement of water molecules indicating presence of specific ethanol hydration shell, as confirmed by results of the RDF curves and coordination number calculations. This behavior, controlled by hydrogen bonding provides a significant contribution to such a conformational transition behavior of the polyelectrolyte chain. The interactions between counter-ions and charges on the PAA chain also influence chain collapse. The underlying origins of polyelectrolyte chain collapse in water–alcohol mixtures are brought out for the first time via explicit MD simulations by this study.     

Comparative structural studies of psychrophilic and mesophilic protein homologues by molecular dynamics simulation

Comparative molecular dynamics simulations of psychrophilic type III antifreeze protein from the North-Atlantic ocean-pout Macrozoarces americanus and its corresponding mesophilic counterpart, the antifreeze-like domain of human sialic acid synthase, have been performed for 10 ns each at five different temperatures. Analyses of trajectories in terms of secondary structure content, solvent accessibility, intramolecular hydrogen bonds and protein–solvent interactions indicate distinct differences in these two proteins. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their growth temperatures. However at higher temperatures psychrophilic protein shows increased overall flexibility than its mesophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are non-overlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar directions of motion of each protein especially at 298 K, 310 K and 373 K. Overall, the psychrophilic protein leads to increased conformational sampling of the phase space than its mesophilic counterpart.Our study may help in elucidating the molecular basis of thermostability of homologous proteins from two organisms living at different temperature conditions. Such an understanding is required for designing efficient proteins with characteristics for a particular application at desired working temperatures.     

Design and synthesis of a novel class of carbonic anhydrase-IX inhibitor 1-(3-(phenyl/4-fluorophenyl)-7-imino-3H-[1,2,3]triazolo[4,5d]pyrimidin 6(7H)yl)urea

Carbonic anhydrase IX (CAIX) is a promising target in cancer therapy especially in the case of hypoxia-induced tumors. The selective inhibition of CA isozymes is a challenging task in drug design and discovery process. Here, we performed fluorescence-binding studies and inhibition assay combined with molecular docking and molecular dynamics (MD) simulation analyses to determine the binding affinity of two synthesized triazolo-pyrimidine urea derived (TPUI and TPUII) compounds with CAIX and CAII. Fluorescence binding results are showing that molecule TPUI has an excellent binding-affinity for CAIX (k_D = 0.048 μM). The TPUII also exhibits an appreciable binding affinity (k_D = 7.52 μM) for CAIX. TPUI selectively inhibits CAIX as compared to TPUII in the 4-NPA assay. Docking studies show that TPUI is spatially well-fitted in the active site cavity of CAIX, and is involve in H-bond interactions with His94, His96, His119, Thr199 and Thr200. MD simulation studies revealed that TPUI efficiently binds to CAIX and essential active site residual interaction is consistent during the entire simulation of 40 ns. These studies suggest that TPUI appeared as novel class of CAIX inhibitor, and may be used as a lead molecule for the development of potent and selective CAIX inhibitor for the hypoxia-induced cancer therapy.     

Using of hydrogen ion-selective poly(vinyl chloride) membrane electrode based on calix[4]arene as thiocyanate ion-selective electrode

A hydrogen ion-selective electrode (ISE) is prepared by using 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetracyanometoxy-calix[4]arene and an investigation about whether it could be used as a thiocyanate ion-selective electrode is made by using its characteristic of becoming thiocyanate sensitive in acidic regions. The electrode of the optimum characteristic has a composition of 1% ionophore, 66% 2-NPOE and 33% poly(vinyl chloride) (PVC). This electrode exhibits a linear response over the range 1.0 × 10⁻¹ to 3.0 × 10⁻⁵ M of thiocyanate with a slope of 52.0 ± 0.2 mV/pSCN. The effects of the pH and the membrane composition are also investigated. The lifetime of the electrode is at least 4 months and its response time is found to be 10–15 s. The selectivity coefficients of some anions are calculated by using mixed solution interference method. Application of the electrode to the potentiometric titration of thiocyanate ion with silver nitrate is reported. There is a good agreement between the results obtained by the proposed electrode and the Mohr method at 95% confidence level.     

Structural study of carboxylesterase from hyperthermophilic bacteria Geobacillus stearothermophilus by molecular dynamics simulation

Carboxylesterases are ubiquitous enzymes with important physiological, industrial and medical applications such as synthesis and hydrolysis of stereo specific compounds, including the metabolic processing of drugs, and antimicrobial agents. Here, we have performed molecular dynamics simulations of carboxylesterase from hyperthermophilic bacterium Geobacillus stearothermophilus (GsEst) for 10 ns each at five different temperatures namely at 300 K, 343 K, 373 K, 473 K and 500 K. Profiles of root mean square fluctuation (RMSF) identify thermostable and thermosensitive regions of GsEst. Unfolding of GsEst initiates at the thermosensitive α-helices and proceeds to the thermostable β-sheets. Five ion-pairs have been identified as critical ion-pairs for thermostability and are maintained stably throughout the higher temperature simulations. A detailed investigation of the active site residues of this enzyme suggests that the geometry of this site is well preserved up to 373 K. Furthermore, the hydrogen bonds between Asp188 and His218 of the active site are stably maintained at higher temperatures imparting stability of this site. Radial distribution functions (RDFs) show similar pattern of solvent ordering and water penetration around active site residues up to 373 K. Principal component analysis suggests that the motion of the entire protein as well as the active site is similar at 300 K, 343 K and 373 K. Our study may help to identify the factors responsible for thermostability of GsEst that may endeavor to design enzymes with enhanced thermostability.     

Perchlorate-selective polymeric membrane electrode based on a cobaloxime as a suitable carrier

A cobaloxime ([chlorobis(dimethylglyoximeato)(triphenylphosphine)] cobalt (III), [Co(dmgH)₂pph₃Cl]) incorporated in a plasticized poly(vinyl chloride) membrane was used to develop a perchlorate-selective electrode. The influence of membrane composition on the electrode response was studied. The electrode exhibits a Nernstian response over the perchlorate concentration range 1.0 × 10⁻⁶ to 1 × 10⁻¹ mol l⁻¹ with a slope of −56.8 ± 0.7 mV per decade of concentration, a detection limit of 8.3 × 10⁻⁷, a wide working pH range (3–10) and a fast response time (<15 s). The electrode shows excellent selectivity towards perchlorate with respect to many common anions. The electrode was used to determine perchlorate in water and human urine.     

Identification of phenoxyacetamide derivatives as novel DOT1L inhibitors via docking screening and molecular dynamics simulation

Dot1-like protein (DOT1L) is a histone methyltransferase that has become a novel and promising target for acute leukemias bearing mixed lineage leukemia (MLL) gene rearrangements. In this study, a hierarchical docking-based virtual screening combined with molecular dynamic (MD) simulation was performed to identify DOT1L inhibitors with novel scaffolds. Consequently, 8 top-ranked hits were eventually identified and were further subjected to MD simulation. It was indicated that all hits could reach equilibrium with DOT1L in the MD simulation and further binding free energy calculations suggested that phenoxyacetamide-derived hits such as L01, L03, L04 and L05 exhibited remarkably higher binding affinity compared to other hits. Among them, L03 showed both the lowest glide score (−12.281) and the most favorable binding free energy (−303.9 +/− 16.5 kJ/mol), thereby making it a promising lead for further optimization.     

Prediction of three-dimensional structures and structural flexibilities of wild-type and mutant cytochrome P450 1A2 using molecular dynamics simulations

In this study, we investigated the effect of genetic polymorphism on the three-dimensional (3D) conformation of cytochrome P450 1A2 (CYP1A2) using molecular dynamics (MD) simulations. CYP1A2, a major drug-metabolizing enzyme among cytochrome P450 enzymes (CYPs), is known to have many variant alleles. The genetic polymorphism of CYP1A2 may cause individual differences in the pharmacokinetics of medicines. By performing 100 ns or longer MD simulations, we investigated the influence of amino acid mutation on the 3D structures and the dynamic properties of proteins. The results show that the static structures were changed by the mutations of amino acid residues, not only near the mutated residues but also in distant portions of the proteins. Moreover, the mutation of only one amino acid was shown to change the structural flexibility of proteins, which may influence the substrate recognition and enzymatic activity. Our results clearly suggest that it is necessary to investigate the dynamic property as well as the static 3D structure for understanding the change of the enzymatic activity of mutant CYP1A2.     

On design and performance evaluation of an integrated SIP and HCoP-B architecture for nested network mobility

This paper proposes a fully-integrated SIP + HCoP-B architecture to provide efficient mobility management of the nested mobile network. It achieves the following merits, which are rare in the literature. First, it reduces network deployment costs by only equipping an integrated SIP mobile server. Second, it supports both SIP-based and non-SIP-based applications. Third, by adopting the analytical model proposed in Mohanty and Akyildiz (2007) [19], mathematical analyses are provided to investigate six performance metrics of SIP + HCoP-B and the other four well-known SIP's over NEMO schemes over the error-prone wireless link. Finally, it is shown that SIP + HCoP-B outperforms these four traditional schemes through intensive simulations.     

Binding and discerning interactions of PTP1B allosteric inhibitors: Novel insights from molecular dynamics simulations

The α7 helix is either disordered or missing in the three co-crystal structures of allosteric inhibitors with protein tyrosine phosphatase 1B (PTP1B). It was modeled in each complex using the open form of PTP1B structure and studied using molecular dynamics (MD) simulations for 25 ns. B-factor analysis of the residues sheds light on its disordered nature in the co-crystal structures. Further, the ability of inhibitors to act as allosteric inhibitor was studied and established using novel hydrogen bond criteria. The MD simulations were utilized to determine the relative importance of electrostatic and hydrophobic component in to the binding of inhibitors. It was revealed that the hydrophobic interactions predominantly drive the molecular recognition of these inhibitors. Per residue energy decomposition analysis attributed dissimilar affinities of three inhibitors to the several hydrogen bonds and non-bonded interactions. Among the secondary structure elements that surround the allosteric site, helices α6, α7 and loop α6–α7 were notorious in providing variable affinities to the inhibitors. A novel hydrophobic pocket lined by the α7 helix residues Val287, Asn289 and Trp291 was identified in the allosteric site. This study provides useful insights for the rational design of high affinity PTP1B allosteric inhibitors.     

Miniature orthogonal optical scanning mirror excited by torsional piezoelectric fiber actuator

A novel optical scanner excited by a torsional piezoelectric fiber actuator is presented. The device consists of a piezoelectric fiber actuator generating torsional and longitudinal vibrations simultaneously and a specially designed metal frame transforming the two vibrations to orthogonal deflections of the mirror. Theoretical and experimental studies were performed on the structure. The changing trends of the vibration modes and resonant frequencies were obtained from finite element simulations. Samples with 1 mm × 1 mm mirrors were fabricated from PZT hollow fibers with a diameter of 1 mm and a stainless steel sheet with a thickness of 50 μm. A horizontal scanning angle of 17.9° and a vertical scanning angle of 2.6° were achieved at 6780 and 10,330 Hz under an applied voltage of 400 V_p–p.