Faithful estimation of dynamics parameters from CPMG relaxation dispersion measurements

Kovrigin, Evgenii L.; Kempf, James; Grey, Michael J.; Loria, J. Patrick

doi:10.1016/j.jmr.2006.01.010

Cited by 81 publications

(84 citation statements)

References 26 publications

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“…[9, 16–21] Statistical analysis of CPMG data has resulted in improved experimental strategies for minimizing data collection times and has helped define the exchange scenarios that can be reliably characterized experimentally. [9, 19] While CPMG relaxation dispersion is widely used in studies of proteins, R1ρ methods are often applied to uniformly 13 C/ 15 N labeled nucleic acids because unwanted carbon-carbon interactions can be more easily suppressed. [15, 22–25] Because higher effective fields can be used in the R1ρ experiment as compared to CPMG, it is also better suited for characterizing faster microsecond exchange processes.…”

Section: Introductionmentioning

confidence: 99%

Evaluating the uncertainty in exchange parameters determined from off-resonance R1ρ relaxation dispersion for systems in fast exchange

Bothe

Stein

Al‐Hashimi

2014

Journal of Magnetic Resonance

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Spin relaxation in the rotating frame (R1ρ) is a powerful NMR technique for characterizing fast microsecond timescale exchange processes directed toward short-lived excited states in biomolecules. At the limit of fast exchange, only kex = k1 + k−1 and Φıx = pGpE(Δω)2 can be determined from R1ρ data limiting the ability to characterize the structure and energetics of the excited state conformation. Here, we use simulations to examine the uncertainty with which exchange parameters can be determined for two state systems in intermediate-to-fast exchange using off-resonance R1ρ relaxation dispersion. R1ρ data computed by solving the Bloch-McConnell equations reveals small but significant asymmetry with respect to offset (R1ρ(ΔΩ) ≠ R1ρ(−ΔΩ)), which is a hallmark of slow-to-intermediate exchange, even under conditions of fast exchange for free precession chemical exchange line broadening (kex/Δω > 10). A grid search analysis combined with bootstrap and Monte-Carlo based statistical approaches for estimating uncertainty in exchange parameters reveals that both the sign and magnitude of Δω can be determined at a useful level of uncertainty for systems in fast exchange (kex/Δω < 10) but that this depends on the uncertainty in the R1ρ data and requires a thorough examination of the multidimensional variation of χ2 as a function of exchange parameters. Results from simulations are complemented by analysis of experimental R1ρ data measured in three nucleic acid systems with exchange processes occurring on the slow (kex/Δω = 0.2; pE = ~ 0.7%), fast (kex/Δω = ~10–16; pE = ~13%) and very fast (kex = 39,000 s−1) chemical shift timescales.

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Section: Introductionmentioning

confidence: 99%

Evaluating the uncertainty in exchange parameters determined from off-resonance R1ρ relaxation dispersion for systems in fast exchange

Bothe

Stein

Al‐Hashimi

2014

Journal of Magnetic Resonance

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“…At such high exchange rates and with data from only one static field, the optimized values of k ex and p minor become highly correlated. 33 To characterize the conformational exchange remaining at 31°C , we recorded off-resonance R 1ρ relaxation dispersion of amide 15 N ( Figure 2). The R 1ρ relaxation dispersion method is suitable for characterizing exchange processes with rates up to 50 000 s −1 , whereas the CPMG relaxation method is limited to exchange processes with rates up to ∼4000 s −1 .…”

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confidence: 99%

A Folded Excited State of Ligand-Free Nuclear Coactivator Binding Domain (NCBD) Underlies Plasticity in Ligand Recognition

et al. 2013

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Intrinsically disordered proteins are renowned for their structural plasticity when they undergo coupled folding and binding to partner proteins. The nuclear coactivator binding domain of CBP is a remarkable example of this adaptability as it folds into two different conformations depending on the binding partner. To understand the role of the conformational ensemble for plasticity in ligand recognition, we investigated the millisecond dynamics of this domain using relaxation dispersion NMR spectroscopy. All NMR signals originating from the domain are broadened, demonstrating that the whole domain experience conformational exchange. The dispersion data can be described by a global two-state exchange process between a ground state and an excited state populated to 8%. The three helices are still folded in the excited state but have a different packing from the ground state; the contact between helices 2 and 3 found in the ground state is broken in the excited state, and a new one is formed between helices 1 and 3. This suggests that while NCBD in the ground state has a structure similar to the complex with the ligand ACTR, the conformation of NCBD in the excited state has some similarity with that of NCBD in complex with the ligand IRF-3. The energy landscape of this domain is thus proposed to resemble the fold-switching proteins that have two coexisting native states, which may serve as a starting point for binding via conformational selection.

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“…This "population shift" (6) has been demonstrated in RNase A by comparing frequency differences between major and minor dynamic states between the free and ligand-bound enzyme (41). This analysis requires very high quality relaxation data because the frequency difference of exchanging conformers is most difficult to obtain in the fast exchange regime (53). To attempt this analysis in ECP, we performed global fittings of residues in each dynamic individual cluster (1 and 2) such that k ex and populations were shared while the frequency difference was individual for each residue.…”

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confidence: 99%

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Gagné

Charest

Morin

et al. 2012

Journal of Biological Chemistry

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Background: It remains unclear whether structural homologues rely on similar concerted motions to promote enzyme function. Results: Ribonuclease homologues display similar, contiguous clustering motions that can be modulated by mutagenesis. Conclusion: Conformational flexibility can be conserved between distant structural homologues. Significance: Controlling dynamics to modulate function has broad implications in protein engineering and allosteric drug design.

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Faithful estimation of dynamics parameters from CPMG relaxation dispersion measurements

Cited by 81 publications

References 26 publications

Evaluating the uncertainty in exchange parameters determined from off-resonance R1ρ relaxation dispersion for systems in fast exchange

Evaluating the uncertainty in exchange parameters determined from off-resonance R1ρ relaxation dispersion for systems in fast exchange

A Folded Excited State of Ligand-Free Nuclear Coactivator Binding Domain (NCBD) Underlies Plasticity in Ligand Recognition

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

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