scholarly journals Rapid measurement of Watson-Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino <sup>1</sup>H CEST

2021 ◽  
Author(s):  
Bei Liu ◽  
Atul Rangadurai ◽  
Honglue Shi ◽  
Hashim Al-Hashimi
Keyword(s):  
High Power ◽  
Dna Sequences ◽  
Dynamic Equilibrium ◽  
Chemical Exchange ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Duplex Dna ◽  
Dna Duplexes ◽  
Base Pairs ◽  
1H Cest

Abstract. In duplex DNA, Watson-Crick A-T and G-C base pairs (bps) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for understanding the role of these non-canonical bps in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Waston-Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino 1H CEST were in very good agreement with counterparts measured using off-resonance 13C/15N spin relaxation in the rotating frame (R1ρ). It is shown that 1H-1H NOE effects which typically introduce artifacts in 1H based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ~10X higher throughput and ~100X lower cost relative to 13C/15N R1ρ, and enabled Hoogsteen chemical exchange measurements undetectable by R1ρ. The results reveal an increased propensity to form Hoogsteen bps near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment opens the door to more comprehensively characterizing Hoogsteen breathing in duplex DNA.

scholarly journals Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino <sup>1</sup>H CEST

2021 ◽  
Vol 2 (2) ◽  
pp. 715-731
Author(s):  
Bei Liu ◽  
Atul Rangadurai ◽  
Honglue Shi ◽  
Hashim M. Al-Hashimi
Keyword(s):  
High Power ◽  
Dna Sequences ◽  
Dynamic Equilibrium ◽  
Chemical Exchange ◽  
Rapid Assessment ◽  
Relaxation Dispersion ◽  
Duplex Dna ◽  
Dna Duplexes ◽  
Base Pairs ◽  
1H Cest

Abstract. In duplex DNA, Watson–Crick A–T and G–C base pairs (bp's) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for identifying potential Hoogsteen hot spots and for understanding the potential roles of Hoogsteen base pairs in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Watson–Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino 1H CEST were in very good agreement with counterparts measured using off-resonance 13C / 15N spin relaxation in the rotating frame (R1ρ). It is shown that 1H–1H NOE effects which typically introduce artifacts in 1H-based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ∼ 10× higher throughput and ∼ 100× lower cost relative to 13C / 15N R1ρ and enabled Hoogsteen chemical exchange measurements undetectable by R1ρ. The results reveal an increased propensity to form Hoogsteen bp's near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment provides a basis for rapidly screening Hoogsteen breathing in duplex DNA, enabling identification of unusual motifs for more in-depth characterization.

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

2019 ◽  
Vol 47 (13) ◽  
pp. 7105-7117 ◽  
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.

scholarly journals A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

2020 ◽  
Vol 74 (12) ◽  
pp. 753-766
Author(s):  
Jan H. Overbeck ◽  
Werner Kremer ◽  
Remco Sprangers
Keyword(s):  
Structural Information ◽  
Chemical Exchange ◽  
Chemical Shifts ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Cold Shock Protein ◽  
X Ray Crystallography ◽  
Kinetic And Thermodynamic Parameters ◽  
Low Costs ◽  
Exchange Parameters

Abstract Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions. Graphic abstract

scholarly journals Probing conformational transitions towards mutagenic Watson–Crick-like G·T mismatches using off-resonance sugar carbon R1ρ relaxation dispersion

2020 ◽  
Vol 74 (8-9) ◽  
pp. 457-471 ◽  
Author(s):  
Atul Rangadurai ◽  
Eric S. Szymanski ◽  
Isaac Kimsey ◽  
Honglue Shi ◽  
Hashim M. Al-Hashimi
Keyword(s):  
Dynamic Equilibrium ◽  
Isotope Effects ◽  
Exchange Process ◽  
Relaxation Dispersion ◽  
Base Pairs ◽  
Carbon And Nitrogen ◽  
Dna And Rna ◽  
Backbone Conformation ◽  
Rna Duplexes ◽  
Dynamics Simulations

Abstract NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson–Crick-like conformations. As Watson–Crick-like G·T mismatches have base pairing geometries similar to Watson–Crick base pairs, we hypothesized that they would mimic Watson–Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3′ and C4′ nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson–Crick conformation and that transitions toward tautomeric and anionic Watson–Crick-like G·T mismatches restore the canonical Watson–Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson–Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T−, and also establish sugar carbons as new non-exchangeable probes of this exchange process.

scholarly journals Visualizing transient dark states by NMR spectroscopy

2015 ◽  
Vol 48 (1) ◽  
pp. 35-116 ◽  
Author(s):  
Nicholas J. Anthis ◽  
G. Marius Clore
Keyword(s):  
Nmr Spectroscopy ◽  
Hydrogen Exchange ◽  
Dynamic Equilibrium ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Paramagnetic Nmr ◽  
Saturation Transfer ◽  
Dark State ◽  
Transient Nature ◽  
High Resolution Analysis

AbstractMyriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.

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

2018 ◽  
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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