relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data

Morin, Sébastien; Linnet, Troels E; Lescanne, Mathilde; Schanda, Paul; Thompson, Gary S.; Tollinger, Martin; Teilum, Kaare; Gagné, Stéphane M.; Marion, Dominique; Griesinger, Christian; Blackledge, Martin; d’Auvergne, Edward J

doi:10.1093/bioinformatics/btz397

Cited by 4 publications

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“…Fits of a two-state exchange model as reported by Meiboom 55 , to the BMCRD data were obtained from the program relax, version 4.0. 56 For the fits shown in Figure 1 we chose five residues for which data of both crystals were available: I23 (only 950 MHz, as no 600 MHz data were available for cubic-PEG-ub) and V26, K27, T55, D58 (600 and 950 MHz). We have investigated how the choice of residues, and the choice of static magnetic field strengths influences the outcome of these fits; we find that using different subgroups of residues in the β-turn region, or including other residues in the fits where available (e.g.…”

Section: Discussionmentioning

confidence: 99%

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Kurauskas

Izmailov

Rogacheva

et al. 2017

Preprint

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Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell relaxation dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with an~3-5°amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.

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Section: Discussionmentioning

confidence: 99%

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Kurauskas

Izmailov

Rogacheva

et al. 2017

Preprint

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“…The 15 N-CPMG relaxation dispersion experiments with NCp7 samples at different concentrations (400 and 100 μM) were recorded at 950 MHz and 35 °C using 11 points with different ν CPMG values. Τhe errors were evaluated using the formula

, ( (accessed on 14 February 2022), relax Manual [ 40 ]) where uncertainties on peak intensities,

and

, were estimated using two repeated points in the CPMG experiments; I 1 (ω1) and I 0 are the peak intensities at a frequency ω 1 in the CPMG series, and I 0 is the experiment performed without the constant time [ 30 ]. Only for the experiments with dilute samples, were the quantity of D2O in samples reduced to 4%, to decrease deuterium isotopic effects that affect the exchange rates [ 41 ].…”

Section: Methodsmentioning

confidence: 99%

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain

Mouhand

Zargarian

Belfetmi

et al. 2022

Viruses

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The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, depending on the processing of Gag by the HIV-1 protease. NCd is constituted of two adjacent zinc knuckles (ZK1 and ZK2), separated by a flexible linker and flanked by disordered regions. Here, conformational equilibria between a major and two minor states were highlighted exclusively in ZK2, by using CPMG and CEST NMR experiments. These minor states appear to be temperature dependent, and their populations are highest at physiological temperature. These minor states are present both in NCp7, the mature form of NCd, and in NCp9 and NCp15, the precursor forms of NCd, with increased populations. The role of these minor states in the targeting of NCd by drugs and its binding properties is discussed.

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“…The R 2,eff was calculated as R 2,eff (ν CPMG ) = (−1/ T ) ln [ I ( ν CPMG )/ I 0 ], where I ( ν CPMG ) and I 0 are the intensities of peaks recorded with and without the CPMG intervals. The 15 N relaxation dispersion profiles for individual residues at both 600 and 850 MHz were fitted to a two‐site exchange model (A↔B), the Carver and Richards equation (CR72), by using the software relax . Successful fit to the CR72 model yields the population‐average transverse relaxation rate ( R 20 ), exchange rate ( k ex ), population of the major state ( p A ), and absolute values of 15 N chemical shift difference between two states (Δ ω N ).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for −35 element recognition by σ₄ chimera proteins and their interactions with PmrA response regulator

et al. 2019

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In class II transcription activation, the transcription factor normally binds to the promoter near the −35 position and contacts the domain 4 of σ factors (σ4) to activate transcription. However, σ4 of σ70 appears to be poorly folded on its own. Here, by fusing σ4 with the RNA polymerase β‐flap‐tip‐helix, we constructed two σ4 chimera proteins, one from σ70 ()σ470normalc and another from σS ()σ4Snormalc of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for −35 element DNA. Determining the crystal structure of σ4Snormalc in complex with −35 element DNA revealed that σ4Snormalc adopts a similar structure as σ4 in the Escherichia coli RNA polymerase σS holoenzyme and recognizes −35 element DNA specifically by several conserved residues from the helix‐turn‐helix motif. By using nuclear magnetic resonance (NMR), σ470normalc was demonstrated to recognize −35 element DNA similar to σ4Snormalc. Carr‐Purcell‐Meiboom‐Gill relaxation dispersion analyses showed that the N‐terminal helix and the β‐flap‐tip‐helix of σ470normalc have a concurrent transient α‐helical structure and DNA binding reduced the slow dynamics on σ470normalc. Finally, only σ470normalc was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of −35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors.

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relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data

Cited by 4 publications

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Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain

Structural basis for −35 element recognition by σ₄ chimera proteins and their interactions with PmrA response regulator

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relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data

Cited by 4 publications

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Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain

Structural basis for −35 element recognition by σ4 chimera proteins and their interactions with PmrA response regulator

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Structural basis for −35 element recognition by σ₄ chimera proteins and their interactions with PmrA response regulator