Native-like mean structure in the unfolded ensemble of small proteins

The nature of the unfolded state plays a great role in our understanding of proteins. However, accurately studying the unfolded state with computer simulation is difficult, due to its complexity and the great deal of sampling required. Using a supercluster of over 10,000 processors we have performed close to 800 micros of molecular dynamics simulation in atomistic detail of the folded and unfolded states of three polypeptides from a range of structural classes: the all-alpha villin headpiece molecule, the beta hairpin tryptophan zipper, and a designed alpha-beta zinc finger mimic. A comparison between the folded and the unfolded ensembles reveals that, even though virtually none of the individual members of the unfolded ensemble exhibits native-like features, the mean unfolded structure (averaged over the entire unfolded ensemble) has a native-like geometry. This suggests several novel implications for protein folding and structure prediction as well as new interpretations for experiments which find structure in ensemble-averaged measurements.     

Carnitine-acylcarnitine translocase. Inhibition by alpha-cyano-4-hydroxycinnamate and evidence for separate identity from the pyruvate transporting system of mitochondria

Some of the known inhibitors of pyruvate transport inhibited the activity of carnitine-acylcarnitine translocase. Their order of effectiveness with millimolar concentration required for 50% inhibition given in parentheses, was: Compound UK-5099 (alpha-cyano-beta-(1-phenylindol-3-yl)acrylate) (0.1); alpha-cyano-4-hydroxycinnamate (0.17); alpha-cyano-3-hydroxycinnamate (1); alpha-cyanocinnamate (1); alpha-fluorocinnamate (7); transcinnamate (10); p-hydroxycinnamate (10); phenylpyruvate (22); p-hydroxyphenylpyruvate (25). Kinetically, the alpha-cyano-4-hydroxycinnamate inhibition was mixed and the p-hydroxyphenylpyruvate inhibition was noncompetitive with respect to external (-)-carnitine. The alpha-cyano-4-hydroxycinnamate inhibition was reversible and resulted from its ability to act as a thiol reagent. In general, alpha-cyanocinnamate and its derivatives inhibit carnitine transport at concentrations 100 to 5000 times as high as those known to pyruvate transport. At millimolar concentrations, alpha-cyano-4-hydroxycinnamate inhibited the mitochondrial transport of molecules other than carnitine as well as the activity of carnitine acyltransferases. Pyruvate and carnitine did not complete for transport into and out of mitochondria. These results establish that transmitochondrial transport mechanisms for carnitine and pyruvate involve different carriers.     

Evidence for the regulatory role of the N-terminal helix of secretory phospholipase A(2) from studies on native and chimeric proteins

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.     

A quantitative structure-activity relationship study on some HIV-1 protease inhibitors using molecular connectivity index

A quantitative structure-activity relationship (QSAR) study has been made on two different series of tetrahydropyrimidinones acting as HIV-1 protease inhibitors. A structural parameter, the first order valence molecular connectivity index ((1)chi(v)), has been used to account for the variation in the activity. The protease inhibition activity as well as the antiviral potency of the compounds are found to be significantly correlated with (1)chi(v) of P(2)/P(2') substituents attached to the two nitrogens N1 and N3, suggesting that substituents containing less electronegative and more saturated atoms, meaning thereby the less polar or more hydrophobic substituents, will be more advantageous. Further, if P(2) and P(2') are dissimilar, the former is found to be more effective than the latter. This difference is attributed to a conformational change in the enzyme that may be more favorable to P(2) binding than to P(2') binding.     

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.