Structure and dynamics of the membrane-bound cytochrome P450 2C9.

The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix

Epigenetic perturbations in the pathogenesis of mustard toxicity; hypothesis and preliminary results

Among the most readily available chemical warfare agents, sulfur mustard (SM), also known as mustard gas, has been the most widely used chemical weapon. SM causes debilitating effects that can leave an exposed individual incapacitated for days to months; therefore delayed SM toxicity is of much greater importance than its ability to cause lethality. Although not fully understood, acute toxicity of SM is related to reactive oxygen and nitrogen species, oxidative stress, DNA damage, poly(ADP-ribose) polymerase (PARP) activation and energy depletion within the affected cell. Therefore several antioxidants and PARP inhibitors show beneficial effects against acute SM toxicity. The delayed toxicity of SM however, currently has no clear mechanistic explanation. One third of the 100,000 Iranian casualties are still suffering from the detrimental effects of SM in spite of the extensive treatment. We, therefore, made an attempt whether epigenetic aberrations may contribute to pathogenesis of mustard poisoning. Preliminary evidence reveals that mechlorethamine (a nitrogen mustard derivative) exposure may not only cause oxidative stress, DNA damage, but epigenetic perturbations as well. Epigenetic refers to the study of changes that influence the phenotype without causing alteration of the genotype. It involves changes in the properties of a cell that are inherited but do not involve a change in DNA sequence. It is now known that in addition to mutations, epimutations contribute to a variety of human diseases. Under light of preliminary results, the current hypothesis will focus on epigenetic regulations to clarify mustard toxicity and the use of drugs to correct possible epigenetic defects

Conformational effects on the Circular Dichroism of Human Carbonic Anhydrase II: a multilevel computational study

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions

Kožna dekontaminacija živčanoga bojnog otrova sarina s apsorpcijskim pripravkom u uvjetima in vivo

Our Institute’s nuclear, biological, and chemical defense research team continuously investigates and develops preparations for skin decontamination against nerve agents. In this in vivo study, we evaluated skin decontamination efficacy against sarin by a synthetic preparation called Mineral Cationic Carrier (MCC®) with known ion exchange, absorption efficacy and bioactive potential. Mice were treated with increasing doses of sarin applied on their skin, and MCC® was administered immediately after contamination. The results showed that decontamination with MCC® could achieve therapeutic efficacy corresponding to 3 x LD50 of percutaneous sarin and call for further research.Istraživački tim NBKO (nuklearno-biološko-kemijske obrane) radi na pronalasku i razvoju pripravka za dekontaminaciju kože od živčanih bojnih otrova. Cilj ovog istraživanja bio je ispitati dekontaminacijska svojstva (adsorpcijska i/ili kemisorpcijska) pripravka MCC® rabeći živčani bojni otrov sarin kao kožni kontaminant u uvjetima in vivo. MCC® je sintetski pripravak koji je biokemijski aktivan i ima ionskoizmjenjivačka i adsorpcijska svojstva. Istraživanje u uvjetima in vivo napravljeno je na miševima aplikacijom rastućih doza sarina na kožu životinje. Pripravak MCC® uporabljen je kao kožni dekontaminant neposredno nakon kožne kontaminacije sarinom. Istraživanja su pokazala da pripravak MCC® posjeduje adsorpcijska svojstva, ujedno važna za dekontaminaciju živčanih bojnih otrova. Eksperimenti u uvjetima in vivo na miševima (NOD-soj) pokazali su da se dekontaminacijom pripravkom MCC® može postići terapijski učinak od 3 LD50 (perkutano, sarin)

Acute and delayed sulfur mustard toxicity; novel mechanisms and future studies

Sulfur mustard (SM), also known as mustard gas, has been the most widely used chemical weapon. The toxicity of SM as an incapacitating agent is of much greater importance than its ability to cause lethality. Acute toxicity of SM is related to reactive oxygen and nitrogen species, DNA damage, poly(ADP-ribose) polymerase activation and energy depletion within the affected cell. Therefore melatonin shows beneficial effects against acute SM toxicity in a variety of manner. It scavenges most of the oxygen- and nitrogen-based reactants, inhibits inducible nitric oxide synthase, repairs DNA damage and restores cellular energy depletion. The delayed toxicity of SM however, currently has no mechanistic explanation. We propose that epigenetic aberrations may be responsible for delayed detrimental effects of mustard poisoning. Epigenetic refers to the study of changes that influence the phenotype without causing alteration of the genotype. It involves changes in the properties of a cell that are inherited but do not involve a change in DNA sequence. It is now known that in addition to genetic mutations, epimutations can also involve in the pathogenesis of a variety of human diseases. Several actions of melatonin are now delineated by epigenetic actions including modulation of histone acetylation and DNA methylation. Future studies are warranted to clarify whether epigenetic mechanisms are involved in pathogenesis of delayed sulfur mustard toxicity and melatonin alleviates delayed toxicity of this warfare agent

Molecular dynamics simulations of a mixed DOPC/DOPG bilayer

We have constructed a mixed dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol bilayer (DOPG) bilayer utilizing MD simulations. The aim was to develop an explicit molecular model of biological membranes as a complementary technique to neutron diffraction studies that are well established within the group. A monolayer was constructed by taking a previously customised PDB file of each molecule and arranging them in a seven rows of ten molecules and duplicated and rotated to form a bilayer. The 140-molecule bilayer contained 98 DOPC molecules and 42 DOPG molecules, in a 7:3 ratio in favour of DOPC. Sodium counter ions were placed near the phosphate moiety of DOPG to counteract the negative charge of DOPG. This was representative of the lipid ratio in a sample used for neutron diffraction. The MD package GROMACS was used for confining the bilayer in a triclinic box, adding Simple Polar Charge water molecules, energy minimization (EM). The bilayer/solvent system was subjected to EM using the steepest descent method to nullify bad contacts and reduce the potential energy of the system. Subsequent MD simulation using an initial NVT (constant number of particles, volume and temperature) for a 20 ps MD run followed by a NPT (constant number of particles, pressure and temperature) was performed. Structural parameters including volume of lipid, area of lipid, order parameter of the fatty acyl carbons and electron density profiles generated by the MD simulation were verified with values obtained from experimental data of DOPC, as there are no comparable experimental data available for the mixed bilayer

Interaction of monotopic membrane enzymes with a lipid bilayer: a coarse-grained MD simulation study.

Monotopic membrane proteins bind tightly to cell membranes but do not generally span the lipid bilayer. Their interactions with lipid bilayers may be studied via coarse-grained molecular dynamics (CG-MD) simulations. Understanding such interactions is important as monotopic enzymes frequently act on hydrophobic substrates, while X-ray structures rarely provide direct information about their interactions with membranes. CG-MD self-assembly simulations enable prediction of the orientation and depth of insertion into a lipid bilayer of a monotopic protein, and also of the interactions of individual protein residues with lipid molecules. The CG-MD method has been evaluated via comparison with extended (>30 ns) atomistic simulations of monoamine oxidase, revealing good agreement between the results of coarse-grained and atomistic simulations. CG-MD simulations have been applied to a set of 11 monotopic proteins for which three-dimensional structures are available. These proteins may be divided into two groups on the basis of the results of the simulations. One group consists of those proteins which are inserted into the lipid bilayer to a limited extent, interacting mainly at the phospholipid-water interface. The second group consists of those which are inserted more deeply into the bilayer. Those monotopic proteins which are inserted more deeply cause significant local perturbation of bilayer properties such as bilayer thickness. Deeper insertion seems to correlate with a greater number of basic residues in the "foot" whereby a monotopic protein interacts with the membrane