title: Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+Titration Analysis for HIV-1 Ribonuclease H Domain.
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 NMR1H-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 MgCl2is 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.
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Ichhuk
authors.initials: I
authors.lastname: Karki
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Martin T
authors.initials: MT
authors.lastname: Christen
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Justin
authors.initials: J
authors.lastname: Spiriti
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Slack
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kanaori
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Zuckerman
authors.affiliation: Department of Structural Biology and ‡Department of Computational and Systems Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.|Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and ⊥Department of Biomolecular Engineering, Kyoto Institute of Technology , Kyoto 606, Japan.
authors.forename: Rieko
authors.identifier: 0000-0002-3418-0922
authors.initials: R
authors.lastname: Ishima
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D008274:Magnesium