Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg(2+) Titration Analysis for HIV-1 Ribonuclease H Domain.
Related Articles Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg(2+) Titration Analysis for HIV-1 Ribonuclease H Domain.
J Phys Chem B. 2016 Dec 15;120(49):12420-12431
Authors: Karki I, Christen MT, Spiriti J, Slack RL, Oda M, Kanaori K, Zuckerman DM, Ishima R
Abstract
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. 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. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR (1)H-(15)N heteronuclear single-quantum coherence spectra upon titration of Mg(2+) into an RNH solution. 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. 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. A possible explanation may be that the titrated MgCl2 is consumed as a salt and interacts with RNH in the absence of NaCl. In addition, our data suggest that Na(+) increases the kinetic rate of the specific Mg(2+) interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.
PMID: 27973819 [PubMed - in process]
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