Common hydrogen bond interactions in diverse phosphoryl transfer active sites.

Phosphoryl transfer reactions figure prominently in energy metabolism, signaling, transport and motility. Prior detailed studies of selected systems have highlighted mechanistic features that distinguish different phosphoryl transfer enzymes. Here, a top-down approach is developed for comparing statistically the active site configurations between populations of diverse structures in the Protein Data Bank, and it reveals patterns of hydrogen bonding that transcend enzyme families. Through analysis of large samples of structures, insights are drawn at a level of detail exceeding the experimental precision of an individual structure. In phosphagen kinases, for example, hydrogen bonds with the O3ß of the nucleotide substrate are revealed as analogous to those in unrelated G proteins. In G proteins and other enzymes, interactions with O3ß have been understood in terms of electrostatic favoring of the transition state. Ground state quantum mechanical calculations on model compounds show that the active site interactions highlighted in our database analysis can affect substrate phosphate charge and bond length, in ways that are consistent with prior experimental observations, by modulating hyperconjugative orbital interactions that weaken the scissile bond. Testing experimentally the inference about the importance of O3ß interactions in phosphagen kinases, mutation of arginine kinase Arg280 decreases kcat, as predicted, with little impact upon KM.

Hyperconjugation-mediated solvent effects in phosphoanhydride bonds.

Density functional theory and natural bond orbital analysis are used to explore the impact of solvent on hyperconjugation in methyl triphosphate, a model for "energy rich" phosphoanhydride bonds, such as found in ATP. As expected, dihedral rotation of a hydroxyl group vicinal to the phosphoanhydride bond reveals that the conformational dependence of the anomeric effect involves modulation of the orbital overlap between the donor and acceptor orbitals. However, a conformational independence was observed in the rotation of a solvent hydrogen bond. As one lone pair orbital rotates away from an optimal antiperiplanar orientation, the overall magnitude of the anomeric effect is compensated approximately by the other lone pair as it becomes more antiperiplanar. Furthermore, solvent modulation of the anomeric effect is not restricted to the antiperiplanar lone pair; hydrogen bonds involving gauche lone pairs also affect the anomeric interaction and the strength of the phosphoanhydride bond. Both gauche and anti solvent hydrogen bonds lengthen nonbridging O-P bonds, increasing the distance between donor and acceptor orbitals and decreasing orbital overlap, which leads to a reduction of the anomeric effect. Solvent effects are additive with greater reduction in the anomeric effect upon increasing water coordination. By controlling the coordination environment of substrates in an active site, kinases, phosphatases, and other enzymes important in metabolism and signaling may have the potential to modulate the stability of individual phosphoanhydride bonds through stereoelectronic effects.

The effect of sample handling on cross sectional HIV incidence testing results.

OBJECTIVE(S): To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays. METHODS: Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4 °C, 25 °C and 37 °C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay). RESULTS: Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4 °C or 25 °C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37 °C prior to testing. CONCLUSIONS: Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4 °C or 25 °C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays.

Effect of sexually transmitted disease (STD) coinfections on performance of three commercially available immunosorbent assays used for detection of herpes simplex virus type 2-specific antibody in men attending Baltimore, Maryland, STD clinics.

Two hundred seventy-nine serum samples from men attending sexually transmitted disease (STD) clinics in Baltimore, Maryland, were tested for herpes simplex virus type 2 (HSV-2)-specific antibody by three immunosorbent glycoprotein G-2-based assays (the Kalon, Focus, and Biokit assays). The results for all samples with positive results were confirmed by Western blotting (91/279; 32.6% HSV-2 seroprevalence). All patients were also tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, human immunodeficiency virus type 1, and hepatitis C virus. The Kalon assay performed very well with samples from this population (90.8% sensitive, 99.4% specific), whereas the Focus assay had a sensitivity (82.6%) much lower than that shown previously. For 19.7% of the samples, the Biokit assay gave an indeterminate result. It was found that the odds of a sample having a Biokit assay indeterminate result compared to that of having a definitive positive or negative results were 3.88 times greater for subjects concurrently infected with N. gonorrhoeae, after the effects of other STDs were controlled for (P = 0.001; 95% confidence interval, 1.78, 8.45). Unfortunately, we were unable to control for HSV-1 infection status in the regression model, which, on the basis of chi(2) analysis, might also affect the clarity of the Biokit test. The recommended index cutoff value of 1.1 for the Focus and Kalon assays was found to be optimal for this population.

Evaluation of a low-cost method, the Guava EasyCD4 assay, to enumerate CD4-positive lymphocyte counts in HIV-infected patients in the United States and Uganda.

OBJECTIVE: To evaluate the EasyCD4 assay, a less expensive method to enumerate CD4+ lymphocytes, in resource-limited settings. DESIGN: Cross-sectional study conducted in the United States and Uganda. METHODS: We compared CD4+ cell counts obtained on replicate samples from HIV-infected patients by the EasyCD4 assay, a microcapillary flow-based system, and by standard flow cytometry or FACSCount with linear regression and the Bland-Altman method. RESULTS: Two hundred eighteen samples were analyzed (77 in the United States and 141 in Uganda). In the United States, mean +/- SD CD4 was 697 +/- 438 cells/microL by standard flow cytometry and 688 +/- 451 cells/microL by EasyCD4. In Uganda, the mean +/- SD CD4 was 335 +/- 331 cells/microL by FACSCount and 340 +/- 327 cells/microL by EasyCD4. The 2 methods were highly correlated (US cohort, r2 = 0.97, slope = 1.0, intercept = -18; Ugandan cohort, r2 = 0.92; slope = 0.95; intercept = 23). The mean differences in CD4 cell counts were 9.0 and -4.6 cells/microL for the US and Ugandan cohorts, respectively, and they were not significant in either cohort. In the Ugandan cohort, sensitivity and specificity of the EasyCD4 for CD4 below 200 cells/microL were 90% and 98%, respectively. Positive predictive value was 96%; negative predictive value was 93%. CONCLUSIONS: Our results suggest that EasyCD4 may be used with high positive and negative predictive value in resource-limited settings.