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The relationship between the conformational properties of 1-34 human parathyroid hormone [hPTH(1-34)] and the oxidation of its methionine residues, Met8 and Met18, by hydrogen peroxide is analyzed as a function of pH by measuring the rates of oxidation and by performing MD simulations with an explicit representation of water molecules. Between pH 4 and pH 8, both Met8 and Met18 have nearly pH independent rates of oxidation, and Met18 is oxidized at a rate that is 90-100% of that of freeMet and 10-20% faster than that of Met8. We also found that average 2SWCNs calculated from MD simulations correlate well to the rates of oxidation of Met8 and Met18. The use of 2SWCNs is based on the mechanism that we proposed, the water-mediated mechanism, in which water molecules stabilize the transition state via specific interactions, but the transfer of protons (acid-catalyzed mechanism) does not play a role [Chu, J. W., and Trout, B. L. (2004) J. Am. Chem. Soc. 126 (3), 900-908]. Only at very low pH values, pH 1 for the oxidation of freeMet, does the acid-catalyzed oxidation mechanism become important. For the oxidation of Met8 and Met18 in hPTH(1-34), the acid-catalyzed mechanism becomes significant at a higher pH value, pH 2, probably due to the proximity of nearby acidic residues to Met8 (Glu4) and Met18 (Glu22). In this study, we have demonstrated that the chemistry of oxidation and the structure of polypeptides can be correlated via a detailed understanding of the reaction mechanism, appropriate sampling of configurational space, and a suitable choice of a structural property, water coordination number.

To understand the connection between the conformation of a protein molecule and the oxidation of its methionine residues, we measured the rates of oxidation of methionine residues by H(2)O(2) in granulocyte colony-stimulating factor (G-CSF) as a function of pH and also studied the structural properties of this protein as a function of pH via molecular dynamics simulations. We found that each of the four methionine groups in G-CSF have significant and different rates of oxidation as a function of pH. Moreover, Met(1), in the unstructured N-terminal region, has a rate of oxidation as low as half that of free methionine. The structural properties of G-CSF as a function of pH are evaluated in terms of properties such as hydrogen bonding, deviations from X-ray structure, helical/helical packing, and the atomic covariance fluctuation matrix of alpha-carbons. We found that dynamics (structural fluctuations) are essential in explaining oxidation and that a static picture, such as that resulting from X-ray data, fails in this regard. Moreover, the simulation results also indicate that the solvent-accessible area, traditionally used to measure solvent accessibility of a protein site, of the sulfur atom of methionine residues does not correlate well with the rate of oxidation. Instead, we identified a structural property, average two-shell water coordination number, that correlates well with measured oxidation rates.

The P-glycoprotein multidrug transporter is a plasma membrane efflux pump for hydrophobic natural products, drugs, and peptides, driven by ATP hydrolysis. Determination of the details of the catalytic cycle of P-glycoprotein is critical if we are to understand the mechanism of drug transport and design ways to inhibit it. It has been proposed that the vanadate-trapped transition state of P-glycoprotein (Pgp x ADP x V(i) x M(2+), where M(2+) is a divalent metal ion) has a very low affinity for drugs compared to resting state protein, thus leading to binding of substrate on the cytoplasmic side of the membrane and release of substrate to the extracellular medium (or the extracellular membrane leaflet). We have used several different fluorescence spectroscopic approaches to show that isolated purified P-glycoprotein, when trapped in a stable transition state with vanadate and either Co(2+)or Mg(2+), binds drugs with high affinity. For vinblastine, colchicine, rhodamine 123, and doxorubicin, the affinity of the vanadate-trapped transition state for drugs was only very slightly (less than 2-fold) lower than the binding affinity of resting state Pgp, whereas for the modulators cyclosporin A and verapamil and the substrate Hoechst 33342, the binding affinity was very similar for the two states. The drug binding affinity of the ADP-bound form of the transporter was also comparable to that of the unoccupied transporter. These results suggest that release of drug from the transporter during the catalytic cycle precedes formation of the transition state.

Glycophorin A was reconstituted into large unilamellar vesicles of egg phosphatidylcholine by detergent dialysis. The observed overall rate of Sendai virus fusion increased approximately 4-fold between 0 and 0.006 mol % glycophorin, roughly proportional to the glycophorin content. However, no further increase in rate was observed at 0.02 mol % glycophorin. Treatment of reassembled glycophorin-liposomes with neuraminidase resulted in a significant decrease in the percent of viral fusion, confirming that the presence of sialic acid residues on glycophorin is essential for its role as a receptor. The sialic acid-containing glycolipid, the ganglioside GD1a, was also incorporated into phosphatidylcholine liposomes, either in addition to or in place of glycophorin A. Comparing, on the basis of sialic acid content, liposomes containing either glycophorin or GD1a, comparable rates and extents of fusion were found. However, on a molar basis glycophorin is much more effective. It was found that the addition of GD1a to glycophorin-containing liposomes only slightly increased the rate of fusion. This was largely due to an increase in the percent of virions capable of fusing.