Compaction of RNA Duplexes in the Cell**

Collauto, Alberto; Bülow, Sören von; Gophane, Dnyaneshwar B.; Saha, Subham; Stelzl, Lukas S.; Hummer, Gerhard; Sigurdsson, Snorri Th.; Prisner, Thomas F.

doi:10.1002/ange.202009800

Cited by 11 publications

(9 citation statements)

References 81 publications

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“…To address this issue, the use of molecular crowders in in vitro studies has been proposed. 78 Our multistate ensemble of the two-domain protein Pin1 resolves its compact and extended states, which we independently observed in our experimental DEER distance distributions. In the absence of ligands, our data support that Pin1 slightly favors the compact state with a ∼70% population.…”

Section: ■ Conclusionsupporting

confidence: 63%

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance

et al. 2021

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Proteins composed of multiple domains allow for structural heterogeneity and interdomain dynamics that may be vital for function. Intradomain structures and dynamics can influence interdomain conformations and vice versa. However, no established structure determination method is currently available that can probe the coupling of these motions. The protein Pin1 contains separate regulatory and catalytic domains that sample “extended” and “compact” states, and ligand binding changes this equilibrium. Ligand binding and interdomain distance have been shown to impact the activity of Pin1, suggesting interdomain allostery. In order to characterize the conformational equilibrium of Pin1, we describe a novel method to model the coupling between intra- and interdomain dynamics at atomic resolution using multistate ensembles. The method uses time-averaged nuclear magnetic resonance (NMR) restraints and double electron–electron resonance (DEER) data that resolve distance distributions. While the intradomain calculation is primarily driven by exact nuclear Overhauser enhancements (eNOEs), J couplings, and residual dipolar couplings (RDCs), the relative domain distribution is driven by paramagnetic relaxation enhancement (PREs), RDCs, interdomain NOEs, and DEER. Our data support a 70:30 population of the compact and extended states in apo Pin1. A multistate ensemble describes these conformations simultaneously, with distinct conformational differences located in the interdomain interface stabilizing the compact or extended states. We also describe correlated conformations between the catalytic site and interdomain interface that may explain allostery driven by interdomain contact.

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Section: ■ Conclusionsupporting

confidence: 63%

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance

et al. 2021

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“…The corresponding measurements are usually executed i) during ~12 hours, ii) at cryogenic temperatures (from 10 K to 60 K, a recent trityl spin-label permitted DEER acquisition at 150 K 746 ), iii) using Q-band (34 GHz) or even the less common W-band (94 GHz) spectrometers, iv) on cellular samples of ~50 µL (10-20 million mammalian cells are enough) containing low-to submicromolar concentrations of doubly spin-labeled proteins/nucleic acids, preferably in deuterated buffers. [391][392][393][394]742,[747][748][749] Hence, the measured distance distribution reports for the ensemble of frozen conformations, independently of the size and location of the studied objects. The distance extraction resolves an ill-posed problem from noisy data, which can bias the distribution shape.…”

Section: Evaluating the Activity Of Oncogenic Mutantsmentioning

confidence: 99%

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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In-cell structural biology aims at extracting structural information about proteins or nucleic acids in their native, cellular environment. This emerging field holds great promises and is already providing new facts and outlooks of interest at both fundamental and applied levels. NMR spectroscopy has important contributions on this stage: it brings information on a broad variety of nuclei at the atomic scale, which ensures its great versatility and uniqueness. Here, we detail the methods, the fundamental knowledge and the applications in biomedical engineering related to in-cell NMR. We finally propose a brief overview of the main other techniques in the field (EPR, smFRET, cryo-ET…) to draw some advisable developments for in-cell NMR. In the era of large-scale screenings and deep learning, both accurate and qualitative experimental evidences are as essential as ever to understand the interior life of cells. In-cell structural biology by NMR spectroscopy can generate such a knowledge, and it does so at the atomic scale. This review is meant to deliver comprehensive but accessible information, with advanced technical details and reflections on the methods, the nature of the results and the future of the field. CONTENTS 5.1.1. In-cell EPR 5.1.2. In-cell FRET microscopy 5.1.3. Mass-spectrometry 5.1.4. Cryo-ET 5.2. The specific benefits of in-cell NMR 5.3. The future technical challenges of in-cell NMR 6. Conclusion Author Information

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“…Although DEER, single-molecule FRET, and luminescence resonance energy transfer have been previously used to study purified and/or reconstituted MsbA ( 12 , 13 , 26 , 29 ), these methods cannot be applied to carry out in vivo experiments with MsbA, because they would require the site-specific and near-complete labeling of engineered cysteines using thiol-reactive reporter dyes in the context of the cell’s proteome. In pioneering studies, in-cell DEER was successfully applied to spin-labeled cytosolic proteins upon electroporation into cells ( 30 , 31 ), spin-labeled RNA injected into oocytes or cells ( 32 ), and bacterial outer membrane proteins directly spin-labeled in the extracellular regions ( 33 ). Recently, we used spin-labeled nanobodies to study membrane transporters in cell-derived membranes ( 34 ).…”

Section: Introductionmentioning

confidence: 99%

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells

et al. 2022

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Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.

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Compaction of RNA Duplexes in the Cell**

Cited by 11 publications

References 81 publications

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells

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