Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain

Ichhuk Karki, Martin T. Christen, Justin Spiriti, Ryan L. Slack, Masayuki Oda, Kenji Kanaori, Daniel Zuckerman, Rieko Ishima

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

This article communicates our study to elucidate the molecular determinants of weak Mg2+ 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 Mg2+ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg2+-titration data. 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 Mg2+ 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 Mg2+ interaction with the active site: (1) weak interaction of Mg2+, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg2+ 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 Mg2+ interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.

Original languageEnglish (US)
Pages (from-to)12420-12431
Number of pages12
JournalJournal of Physical Chemistry B
Volume120
Issue number49
DOIs
StatePublished - Dec 15 2016

Fingerprint

Human Immunodeficiency Virus Ribonuclease H
Ribonuclease H
human immunodeficiency virus
Titration
titration
HIV-1
Nuclear magnetic resonance
nuclear magnetic resonance
proteins
Proteins
interactions
Catalytic Domain
salts
Salts
data simulation
Magnesium Chloride
determinants
amides
Chemical shift
chemical equilibrium

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra : A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain. / Karki, Ichhuk; Christen, Martin T.; Spiriti, Justin; Slack, Ryan L.; Oda, Masayuki; Kanaori, Kenji; Zuckerman, Daniel; Ishima, Rieko.

In: Journal of Physical Chemistry B, Vol. 120, No. 49, 15.12.2016, p. 12420-12431.

Research output: Contribution to journalArticle

Karki, Ichhuk ; Christen, Martin T. ; Spiriti, Justin ; Slack, Ryan L. ; Oda, Masayuki ; Kanaori, Kenji ; Zuckerman, Daniel ; Ishima, Rieko. / Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra : A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain. In: Journal of Physical Chemistry B. 2016 ; Vol. 120, No. 49. pp. 12420-12431.
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AB - This article communicates our study to elucidate the molecular determinants of weak Mg2+ 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 Mg2+ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg2+-titration data. 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 Mg2+ 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 Mg2+ interaction with the active site: (1) weak interaction of Mg2+, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg2+ 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 Mg2+ interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.

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