![]() ![]() Note the bottom region includes Cl − density surrounded by Na + density. In panel B, there is a region of increased Na + density near the substrate handle region. The isosurfaces are at an isovalue of 1.7 × 10 −4 (Å −3), corresponding to a salt concentration of approximately 282 mM (described in the Results). In (B), residues at the RNH substrate-handle region (residues 503 to 527, ) are highlighted as green ribbons with stick representation of the side chains. In (A), red highlights 20 selected residues, for which chemical shift changes at all the Mg 2+ concentrations were detected without signal overlap and those at 80 mM Mg 2+ were one-standard deviation larger than the average values among all the residues (described in the Results). Ribbon representations of the structure of the RNH domain and the metal binding site (yellow side chains), (A) alone and (B) with isosurface overlay showing ion densities of Na + (red) and Cl − (blue) calculated using MD trajectories. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein. In addition, our data suggest that Na + increases the kinetic rate of the specific Mg 2+ interaction at the active site of RNH. A possible explanation may be that the titrated MgCl 2 is consumed as a salt and interacts with RNH in the absence of NaCl. Careful analysis of these three sets of titration data, along with molecular dynamics simulation data of RNH with Na + and Cl - ions, demonstrates two characteristic phenomena distinct from the specific Mg 2+ interaction with the active site: (1) weak interaction of Mg 2+, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg 2+ affinity in the absence of NaCl compared to that in the presence of 50 mM NaCl. We performed the titration under three different conditions: (1) in the absence of NaCl, (2) in the presence of 50 mM NaCl, and (3) at a constant 160 mM Cl - concentration. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR 1H- 15N heteronuclear single-quantum coherence spectra upon titration of Mg 2+ into an RNH solution. As the interaction is weak (a ligand-dissociation constant >1 mM), nonspecific Mg 2+ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg 2+-titration data. The complete sets of 1H and 13C NMR data for pregnenolone, pregnenolone 3-acetate, and 16-dehydropregnenolone serve as reference standards.This article communicates our study to elucidate the molecular determinants of weak Mg 2+ interaction with the ribonuclease H (RNH) domain of HIV-1 reverse transcriptase in solution. Accurate assignments were similarly derived for signals from C-2, C-7, C-8, and C-21 of 1 and 3, and C-15 and C-16 of 1, 2, and 3. This now corrects the earlier conflicts among assignments reported for 13C signals of 1 and 3. Assignments based on 2D COSY spectra were confirmed by correlation peaks from HSQC. Low intensity cross peaks were seen for H-4 coupled with H-6 and H-7, and also H-16 (quasi-axial) with H-14 of 1 and 3. We demonstrated that NUS improved sensitivity compared to uniform sampling (US). In this study, we evaluated the utility of NUS 1 H- 13 C heteronuclear single quantum coherence (HSQC) for semi-quantitative metabolomics. ![]() (SOFAST) technique coupled to heteronuclear multiple quantum correlation (HMQC) along with nonlinear sampling (NUS) in urine and serum samples. Similar cross peaks were observed for H-17 with H-21. Despite these advantages, the technique is not widely applied to metabolomics. However owing to poorer natural abundance and gyromagnetic ratio of 13 C, the acquisition time for 2D 1 H-13 C heteronuclear single quantum coherence spectroscopy (HSQC) is long. Assignments were based on high intensity cross peaks from long-range coupling by H-18 with H-17 and H-12 (axial). The narrow band of overlapping signals from H-7, H-2, and H-4 was resolved by exploiting three-bond coupling in the 2D COSY spectra and heteronuclear correlation. Assignments for signals from 1H and 13C in the NMR spectra of pregnenolone (1), 16-dehydropregnenolone (2), and the 3-acetate of 1 (3) were validated by two-dimensional correlated spectroscopy (2D COSY) and heteronuclear single quantum coherence (HSQC). ![]()
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