Molecular dynamics simulation of human prion protein including both N-linked oligosaccharides and the GPI anchor

Although glycosylation appears to protect prion protein (PrPC) from the conformational transition to the disease-associated scrapie form (PrPSc), available NMR structures are for non-glycosylated PrPC, only, To investigate the influence of both the two N-linked glycans, Asn181 and Asn197, and of the GPI anchor attached to Ser230, on the structural, dynamical and electrostatic behavior of PrP, we have undertaken molecular dynamics simulations on the C-terminal region of human prion protein HuPrP(90-230), with and without the three glycans, The simulations used the AMBER94 force field in a periodic box model with explicit water molecules, considering all long-range electrostatic interactions, The results suggest the structured part of the protein, HuPrP(127-227) is stabilized overall from addition of the glycans, specifically by extensions of Helix-B and Helix-C and reduced flexibility of the linking turn containing Asn197, although some regions such as residues in the turn (165-170) between Strand-B and Helix-B have increased flexibility. The stabilization appears indirect, by reducing the mobility of the surrounding water molecules, and not from specific interactions such as H bonds or ion pairs, The results are consistent with glycosylation at Asn197 having a stabilizing role, while that at Asn181, in a region with already stable secondary structure, having a more functional role, in agreement with literature suggestions, Due to three negatively charged SiaLe(x) groups per N-glycan, the surface electrostatic properties change to a negative electrostatic field covering most of the C-terminal part, including the surface of Helix-B and Helix-C, while the positively charged N-terminal part PrP(90-126) of undefined structure creates a positive potential, The unusual hydrophilic Helix-A (144-152) is not covered by either of these dominant electrostatic fields, and modeling shows it could readily dimerize in anti parallel fashion,In combination with separate simulations of the GPI anchor in membrane model, the results show the GPI anchor is highly flexible and would maintain the protein at a distance between 9 and 13 Angstrom from the membrane surface, with little influence on its structure or orientational freedom

Molecular dynamics simulations of the docking of substituted N5-deazapterins to dihydrofolate reductase

Orientations of the deazapterin ring and the conformational preferences of groups appended to the deazapterin ring in a set of 8-substituted deazapterin cations docked into the dihydrofolate reductase (DHFR) binding site have been investigated using a methodology based on the simulated annealing technique within molecular dynamics (MD) simulations. Of five possible binding pockets for the 8-substituents, identified from a preliminary manual docking study, one has been definitively eliminated after an analysis of MD trajectories, while another remains uncertain. Using a new method based on standard thermodynamic cycles and a linear approximation of polar and non-polar free energy contributions from MD averages, binding affinities of the different ligands in each binding site have been correlated with experimental dissociation constants. The study has provided insights into structure-activity relation-ships for use in the design of modified inhibitors of DHFR

Comparison of electrostatic potential around proteins calculated from Amber and AM1 charges: application to mutants of prion protein

On the basis of arguments of complementary fit of shape and charge polarity or hydrophobicity, molecular electrostatic potentials (MEPs) around proteins are commonly used to deduce likely sites for interaction with ligands or other proteins, including for variations such as mutations. But protein MEPs calculated classically from fixed force field descriptions, including those with implicit solvent models such as in Delphi, do not allow for repolarization of protein residues within the protein system; hence, their representations are likely to be variably inaccurate. Linear-scaling methods now allow calculation of MEPs quantum mechanically for systems as large as proteins, and can account for polarization explicitly. Here we compare MEPs derived from AM1 charge distributions calculated by Mopac2000 with those from the classical Amber force field. Our models are mutants of prion protein (PrP), a protein with an unusually high number of charged residues. The results demonstrate that static point charges, as used in most current force fields, cannot reproduce the MEP of macromolecules. Also, it is not sufficient to account for the influence of nearby atoms connected by chemical bonds; the influence of nearby atoms in space is at least as important. Thus, further progress in the accuracy and wider applicability of force fields requires proper accounting for polarization. Mopac2000 calculations can provide the necessary data for checking new force fields and/or parameter fitting

Acute oxygen supplementation restores markers of hepatocyte energy status and hypoxia in cirrhotic rats

The oxygen limitation hypothesis states that hepatocyte hypoxia is the mechanism determining metabolic restriction in the cirrhotic liver. Therefore we studied markers of hepatocyte energy state and cellular hypoxia in livers of normal and cirrhotic rats before and after oxygen supplementation. Rats with carbon tetrachloride-induced cirrhosis and procedural control rats were exposed to either room air or a hyperoxic gas mixture for 1 h immediately before freeze clamping and perchloric acid extraction of liver tissue. Extracts were assessed by P-31 NMR and enzymatic assays. Livers from cirrhotic rats breathing room air showed a reduced ratio of ATP/ADP, an increased ratio of inorganic phosphate/ATP, and a trend toward an increased ratio of lactate/pyruvate compared with procedural control livers (ATP/ADP 1.73 +/- 0.35 versus 2.68 +/- 0.61, P < .05; P-i/ATP 2.74 +/- 0.48 versus 1.56 +/- 0.26, P < .05; lactate/pyruvate 29.3 +/- 6.4 versus 22.5 +/- 7.4, P = .18). After supplementation with oxygen for 1 h, these ratios in cirrhotic livers approached control values. A variety of other metabolic markers affected by cirrhosis showed variable trends toward normal in response to oxygen supplementation, whereas minor trends toward an increase in ATP levels in control animals suggest the possibility of marginal oxygen limitation in normal livers. The data are consistent with the hypothesis that hepatocytes in cirrhotic livers have normal metabolic capacity but are constrained by a deficit in oxygen supply. Interventions aimed at increasing oxygen supply to the liver may have both short- and long-term therapeutic value in the management of cirrhosis