Recent Developments in 15N NMR Relaxation Studies that Probe Protein Backbone Dynamics

Ishima, Rieko

doi:10.1007/128_2011_212

Cited by 22 publications

(31 citation statements)

References 126 publications

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“…Each spectrum was collected with 64 scans and the same measurements were repeated three times. The R 2 dispersion profiles of non‐overlapping peaks were globally fitted to the equation derived for a two‐state conformational exchange model (A ⇄ B) under the fast conformational exchange regime :

R_{2, eff} = R_{2, 0} + R_{normalex} = R_{2, 0} + {normalΦ}_{normalex} false[1 - (4 / k_{normalex} {normalτ}_{normalCP}) tanh (k_{normalex} {normalτ}_{normalCP} / 4) false] / k_{normalex}

where R 2,0 is the population‐average intrinsic relaxation rate and k ex (= k AB + k BA ) is the rate of exchange between the states. Φ ex is a scaling factor and expressed as p A p B Δω 2 , where p A and p B are the fractional populations of the A and B states, respectively, and Δω is the chemical shift difference between these two states.…”

Section: Methodsmentioning

confidence: 99%

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Functional conformer of c‐Myb DNA‐binding domain revealed by variable temperature studies

et al. 2015

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The conformational fluctuation in the minimum DNA-binding domain of cMyb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70°C, with a transition temperature of approximately 50°C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both 1 H one-dimensional and 15 N/ 1 H two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity-filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37°C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37°C, which is more amenable to DNA-binding.

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R_{2, eff} = R_{2, 0} + R_{normalex} = R_{2, 0} + {normalΦ}_{normalex} false[1 - (4 / k_{normalex} {normalτ}_{normalCP}) tanh (k_{normalex} {normalτ}_{normalCP} / 4) false] / k_{normalex}

Section: Methodsmentioning

confidence: 99%

“…Each spectrum was collected with 64 scans and the same measurements were repeated three times. The R 2 dispersion profiles of non-overlapping peaks were globally fitted to the equation derived for a two-state conformational exchange model (A ⇄ B) under the fast conformational exchange regime [26,27]:…”

Section: Nmr Measurementsmentioning

confidence: 99%

Functional conformer of c‐Myb DNA‐binding domain revealed by variable temperature studies

et al. 2015

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“…NMR experiments to determine 15 N transverse relaxation rate (R 2 ), 15 N longitudinal relaxation rate (R 1 ), and 15 N- 1 H heteronuclear Nuclear Overhauser Enhancement (hNOE) were performed using Bruker Avance 600 MHz NMR spectrometers equipped with a cryogenic probeat 20°C for the WT and flap mutant 13, 47 . In the R 1 and R 2 experiments, spectra were recorded with 7 relaxation delay points: 0, 8, 16, 24, 32, 48, and 64 ms for R 2 , and 0, 50, 100, 200, 300, 500, and 800 ms for R 1 .…”

Section: Methodsmentioning

confidence: 99%

Differential Flap Dynamics in Wild-Type and a Drug Resistant Variant of HIV-1 Protease Revealed by Molecular Dynamics and NMR Relaxation

Cai

Yilmaz

Myint

et al. 2012

J. Chem. Theory Comput.

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In the rapidly evolving disease of HIV drug resistance readily emerges, nullifying the effectiveness of therapy. Drug resistance has been extensively studied in HIV-1 protease where resistance occurs when the balance between enzyme inhibition and substrate recognition and turn-over is perturbed to favor catalytic activity. Mutations which confer drug resistance can impact the dynamics and structure of both the bound and unbound forms of the enzyme. Flap+ is a multi-drug-resistant variant of HIV-1 protease with a combination of mutations at the edge of the active site, within the active site, and in the flaps (L10I, G48V, I54V, V82A). The impact of these mutations on the dynamics in the unliganded form in comparison with the wild-type protease was elucidated with Molecular Dynamic simulations and NMR relaxation experiments. The comparative analyses from both methods concur in showing that the enzyme’s dynamics are impacted by the drug resistance mutations in Flap+ protease. These alterations in the enzyme dynamics, particularly within the flaps, likely modulate the balance between substrate turn-over and drug binding, thereby conferring drug resistance.

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“…Several methods have been developed for analyzing protein dynamics [51], [64], [65]. Here, we used the TENSOR2 program [48] and a suit of Mathematica notebooks provided by Spyracopoulos's lab [49] to analyze 15 N relaxation data.…”

Section: Methodsmentioning

confidence: 99%

Solution Structural Analysis of the Single-Domain Parvulin TbPin1

Sun

Peng

et al. 2012

PLoS ONE

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BackgroundPin1-type parvulins are phosphorylation-dependent peptidyl-prolyl cis-trans isomerases. Their functions have been widely reported to be involved in a variety of cellular responses or processes, such as cell division, transcription, and apoptosis, as well as in human diseases including Alzheimer's disease and cancers. TbPin1 was identified as a novel class of Pin1-type parvulins from Trypanosoma brucei, containing a unique PPIase domain, which can catalyze the isomerization of phosphorylated Ser/Thr-Pro peptide bond.Methodology/Principal FindingsWe determined the solution structure of TbPin1 and performed 15N relaxation measurements to analyze its backbone dynamics using multi-dimensional heteronuclear NMR spectroscopy. The average RMSD values of the 20 lowest energy structures are 0.50±0.05 Å for backbone heavy atoms and 0.85±0.08 Å for all heavy atoms. TbPin1 adopts the typical catalytic tertiary structure of Pin1-type parvulins, which comprises a globular fold with a four-stranded anti-parallel β-sheet core surrounded by three α-helices and one 310-helix. The global structure of TbPin1 is relatively rigid except the active site. The 2D EXSY spectra illustrate that TbPin1 possesses a phosphorylation-dependent PPIase activity. The binding sites of TbPin1 for a phosphorylated peptide substrate {SSYFSG[p]TPLEDDSD} were determined by the chemical shift perturbation approach. Residues Ser15, Arg18, Asn19, Val21, Ser22, Val32, Gly66, Ser67, Met83, Asp105 and Gly107 are involved in substantial contact with the substrate.Conclusions/SignificanceThe solution structure of TbPin1 and the binding sites of the phosphorylated peptide substrate on TbPin1 were determined. The work is helpful for further understanding the molecular basis of the substrate specificity for Pin1-type parvulin family and enzyme catalysis.

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Recent Developments in 15N NMR Relaxation Studies that Probe Protein Backbone Dynamics

Cited by 22 publications

References 126 publications

Functional conformer of c‐Myb DNA‐binding domain revealed by variable temperature studies

Functional conformer of c‐Myb DNA‐binding domain revealed by variable temperature studies

Differential Flap Dynamics in Wild-Type and a Drug Resistant Variant of HIV-1 Protease Revealed by Molecular Dynamics and NMR Relaxation

Solution Structural Analysis of the Single-Domain Parvulin TbPin1

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