Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain.
J Phys Chem B
; 120(49): 12420-12431, 2016 12 15.
Article
in En
| MEDLINE
| ID: mdl-27973819
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
HIV-1
/
Ribonuclease H
/
HIV Reverse Transcriptase
/
Magnesium
Type of study:
Prognostic_studies
Language:
En
Journal:
J Phys Chem B
Journal subject:
QUIMICA
Year:
2016
Document type:
Article
Affiliation country:
Estados Unidos