Improved pKa calculations through flexibility based sampling of a water-dominated interaction scheme

Ionizable groups play critical roles in biological processes. Computation of pK(a)s is complicated by model approximations and multiple conformations. Calculated and experimental pK(a)s are compared for relatively inflexible active-site side chains, to develop an empirical model for hydration entropy changes upon charge burial. The modification is found to be generally small, but large for cysteine, consistent with small molecule ionization data and with partial charge distributions in ionized and neutral forms. The hydration model predicts significant entropic contributions for ionizable residue burial, demonstrated for components in the pyruvate dehydrogenase complex. Conformational relaxation in a pH-titration is estimated with a mean-field assessment of maximal side chain solvent accessibility. All ionizable residues interact within a low protein dielectric finite difference (FD) scheme, and more flexible groups also access water-mediated Debye-Hückel (DH) interactions. The DH method tends to match overall pH-dependent stability, while FD can be more accurate for active-site groups. Tolerance for side chain rotamer packing is varied, defining access to DH interactions, and the best fit with experimental pK(a)s obtained. The new (FD/DH) method provides a fast computational framework for making the distinction between buried and solvent-accessible groups that has been qualitatively apparent from previous work, and pK(a) calculations are significantly improved for a mixed set of ionizable residues. Its effectiveness is also demonstrated with computation of the pH-dependence of electrostatic energy, recovering favorable contributions to folded state stability and, in relation to structural genomics, with substantial improvement (reduction of false positives) in active-site identification by electrostatic strain.     

Prediction of pKa and redox properties in the thioredoxin superfamily

Electrostatic interactions play important roles in diverse biological phenomena controlling the function of many proteins. Polar molecules can be studied with the FDPB method solving the Poisson-Boltzmann equation on a finite difference grid. A method for the prediction of pK(a)s and redox potentials in the thioredoxin superfamily is introduced. The results are compared with experimental pK(a) data where available, and predictions are made for members lacking such data. Studying CxxC motif variation in the context of different background structures permits analysis of contributions to cysteine DeltapK(a)s. The motif itself and the overall framework regulate pK(a) variation. The reported method includes generation of multiple side-chain rotamers for the CxxC motif and is an effective predictive tool for functional pK(a) variation across the superfamily. Redox potential follows the trend in cysteine pK(a) variation, but some residual discrepancy indicates that a pH-independent factor plays a role in determining redox potentials for at least some members of the superfamily. A possible molecular basis for this feature is discussed.     

Thapsigargin and EGTA inhibit endothelin-1-induced glucose transport

We have previously demonstrated that ET-1 may enhance glucose transport in 3T3-L1 adipocytes, secondarily to its stimulatory effect on GLUT1 gene expression by a mitogen-activated protein kinase (MAPK)-dependent pathway. In the present study, we further tested the involvement of Ca²⁺ in glucose uptake in response to ET-1. Among a variety of Ca²⁺-related agents tested, EGTA and thapsigargin were found to suppress both the glucose uptake and intracellular Ca²⁺ mobilization induced by ET-1, as determined by Fura-2 analysis. However, a phospholipase C inhibitor, U73122, also eliminated the intracellular calcium mobilization induced by ET-1, but had no effect on ET-1-stimulated glucose uptake. The finding that neither EGTA nor thapsigargin had any influence on ET-1-induced MAPK activation implies that some mechanism downstream of MAPK activation is involved. Further investigation showed that both agents exerted global inhibitory effects on protein and RNA syntheses. Since both thapsigargin and EGTA may deplete endoplasmic reticulum (ER) Ca²⁺ stores, our results suggest that (1) ET-1-induced glucose transport is independent of ET-1's effect on Ca²⁺ mobilization and (2) depletion of ER Ca²⁺ stores per se may interfere with ET-1's effect on GLUT1 expression.     

Aryl tetrahydropyridine inhibitors of farnesyltransferase: bioavailable analogues with improved cellular potency

Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered bioavailable aryl tetrahydropyridines that are potent in cell culture. The design, synthesis, SAR and biological properties of these compounds will be discussed.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

Feeding habits of guanacos<Emphasis Type="Italic">Lama guanicoe</Emphasis> in the high Andes of north-central Chile

The guanacoLama guanicoe Muller, 1776 has a wide distribution along the Andes and Patagonia. We studied the feeding behaviour of a guanaco population that lives over 4100 m altitude in the Andes of north-central Chile. By contrasting the diet of guanacos during a dry year with that of a wet year and comparing it with the plant availabilities in the field, we tested the hypothesis that the guanaco is a generalist herbivore. We predicted that under such extreme habitat conditions guanacos should consume whatever plant species are available in the environment, especially in a dry year, when vegetation is scarcer. In addition, we compared its diet at three different age classes. We estimated the diet through the microhistological analysis of plant remains found in guanaco pellets collected during January of 1997 (ie after a dry year) and 1998 (ie after a wet year; 41 vs 495 mm, respectively). Then, we computed dietary preferences, food niche-breadth, and food-niche overlap between years and among age classes. Vegetation cover and plant species richness were higher during the wet than during the dry year. The most common plants in the environment were perennial graminoids and legumes. Contrary to our prediction, the guanaco preferred a few plant species, showing a relatively narrow diet breadth that changed little between years differing in plant abundances. The diet proportions differed among the three age classes, however. Our data indicate that at least in this high-elevation population, guanacos are selective and non-opportunistic herbivores. This specialized feeding behaviour is puzzling given the energetic demands of living in a harsh environment with low availabilities of resources. The hypothesis that this is due to the lower palatability of the plants not eaten, remains to be tested.