Comprehensive analysis of NMR data using advanced line shape fitting

Niklasson, Markus; Otten, Renee; Ahlner, Alexandra; Andrésen, Cecilia; Schlagnitweit, Judith; Petzold, Katja; Lundström, Patrik

doi:10.1007/s10858-017-0141-6

Cited by 65 publications

(58 citation statements)

References 34 publications

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“…Dissociation constants for Suc-AFPF-pNA at 10 °C were obtained by titrating the peptide (final concentrations: 0, 1, 3, 5, 9 mM) into a solution of 0.25 mM CypA. Line-shape fitting was performed using PINT 53 , 54 to obtained cross peak positions in the individual spectra. The combined chemical shift difference Δ δ was calculated according to Eq.…”

Section: Methodsmentioning

confidence: 99%

“…Line-shape fitting was performed using PINT 53 , 54 and the obtained cross peak volumes were used to calculate the values of R 2,eff . Error estimation in the experimental data using the four duplicate data points was performed as described earlier by Mulder et al 60 For ease of comparison, the values of R 2,inf were calculated by taking the average of the R 2,eff at the three highest υ CPMG values (i.e., 900, 950, and 1000 Hz) and normalized to the lowest temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Error estimation in the experimental data using the four duplicate data points was performed as described earlier by Mulder et al 60 For ease of comparison, the values of R 2,inf were calculated by taking the average of the R 2,eff at the three highest υ CPMG values (i.e., 900, 950, and 1000 Hz) and normalized to the lowest temperature. Data sets for each mutant at the different temperatures were fitted using PINT 53 , 54 to analytical equations for two-site exchange 61 – 63 and the F -test was used to determine whether a residue experiences exchange. If at any of the temperatures the exchange is determined to be significant ( p < 0.05) for a residue, it is included in the respective supplementary data.…”

Section: Methodsmentioning

confidence: 99%

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Rescue of conformational dynamics in enzyme catalysis by directed evolution

et al. 2018

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Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes. Protein dynamics is recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we use directed evolution on an impaired mutant of the proline isomerase CypA and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen is correlated with an accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles. Our data provide a glimpse of an evolutionary trajectory and show how subtle changes can fine-tune enzyme function.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Rescue of conformational dynamics in enzyme catalysis by directed evolution

et al. 2018

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“…Backbone resonance assignment of 2 H-, 15 All spectra were processed using NMRPipe (39) and analysed using CARA (http://cara.nmr.ch), CcpNmr Analysis (40), or Sparky (41). Peak fitting, error estimation and exponential fitting for the relaxation experiments was done using PINT (42,43). The experimental rotational correlation times were calculated according to Equation 1,…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Structure and dynamics of the quaternaryhunchbackmRNA translation repression complex

Macošek

Simon

Linse

et al. 2020

Preprint

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A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

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“…Duplicate data points were recorded for the 16 ms relaxation delay (36). PINT (38,39) was used for peak integration and data fitting to derive spin relaxation parameters.…”

Section: Nmr Relaxationmentioning

confidence: 99%

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Jagtap¹,

Müller

Masiewicz³

et al. 2018

Preprint

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Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila.Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies.MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs.NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

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Comprehensive analysis of NMR data using advanced line shape fitting

Cited by 65 publications

References 34 publications

Rescue of conformational dynamics in enzyme catalysis by directed evolution

Rescue of conformational dynamics in enzyme catalysis by directed evolution

Structure and dynamics of the quaternaryhunchbackmRNA translation repression complex

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

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