NMR Relaxation Dispersion Methods for the Structural and Dynamic Analysis of Quickly Interconverting, Low-Populated Conformational Substates

Nuclear magnetic resonance relaxation dispersion (rd) experiments provide kinetics and thermodynamics information of molecules undergoing conformational exchange. Rd experiments often use a Carr-Purcell-Meiboom-Gill (CPMG) pulse train equally separated by a spin-state selective inversion element (U-element). Even with measurement parameters carefully set, however, parts of 1H–15N correlations sometimes exhibit large artifacts that may hamper the subsequent analyses. We analyzed such artifacts with a combination of NMR measurements and simulation. We found that particularly the lowest CPMG frequency (νcpmg) can also introduce large artifacts into amide 1H–15N and aromatic 1H–13C correlations whose 15N/13C resonances are very close to the carrier frequencies. The simulation showed that the off-resonance effects and miscalibration of the CPMG π pulses generate artifact maxima at resonance offsets of even and odd multiples of νcpmg, respectively. We demonstrate that a method once introduced into the rd experiments for molecules having residual dipolar coupling significantly reduces artifacts. In the method the 15N/13C π pulse phase in the U-element is chosen between x and y. We show that the correctly adjusted sequence is tolerant to miscalibration of the CPMG π pulse power as large as ±10% for most amide 15N and aromatic 13C resonances of proteins.

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Accelerating 2D NMR relaxation dispersion experiments using iterated maps

Journal of Biomolecular NMR ◽

10.1007/s10858-019-00263-3 ◽

2019 ◽

Vol 73 (10-11) ◽

pp. 561-576 ◽

Cited By ~ 3

Author(s):

Jared Rovny ◽

Robert L. Blum ◽

J. Patrick Loria ◽

Sean E. Barrett

Keyword(s):

Nmr Relaxation ◽

2D Nmr ◽

Relaxation Dispersion ◽

Iterated Maps

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Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev-binding site

Nucleic Acids Research ◽

10.1093/nar/gkz498 ◽

2019 ◽

Vol 47 (13) ◽

pp. 7105-7117 ◽

Cited By ~ 17

Author(s):

Chia-Chieh Chu ◽

Raphael Plangger ◽

Christoph Kreutz ◽

Hashim M Al-Hashimi

Keyword(s):

Binding Site ◽

Nuclear Export ◽

Dynamic Equilibrium ◽

Nmr Relaxation ◽

Conformational Flexibility ◽

Relaxation Dispersion ◽

Conformational States ◽

Rev Response Element ◽

Local Secondary Structure ◽

Hiv 1

AbstractThe HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.

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High-field liquid state NMR hyperpolarization: a combined DNP/NMRD approach

Physical Chemistry Chemical Physics ◽

10.1039/c4cp02451f ◽

2014 ◽

Vol 16 (35) ◽

pp. 18781-18787 ◽

Cited By ~ 25

Author(s):

Petr Neugebauer ◽

Jan G. Krummenacker ◽

Vasyl P. Denysenkov ◽

Christina Helmling ◽

Claudio Luchinat ◽

...

Keyword(s):

Magnetic Field ◽

High Magnetic Field ◽

Dynamic Nuclear Polarization ◽

Liquid State ◽

High Field ◽

Nuclear Polarization ◽

Nmr Relaxation ◽

Relaxation Dispersion ◽

Liquid Solutions ◽

High Fields

Dynamic nuclear polarization and NMR relaxation dispersion measurements have been performed on liquid solutions of TEMPOL radicals in solvents with different viscosities at a high magnetic field of 9.2 T. The results indicate that fast dynamics significantly contribute to DNP enhancements at high fields.

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