In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

Hänsel, Robert; Luh, Laura M.; Corbeski, Ivan; Trantı́rek, Lukáš; Dötsch, Volker

doi:10.1002/anie.201311320

Cited by 94 publications

(76 citation statements)

References 118 publications

(201 reference statements)

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“…5 In-cell NMR experiments are limited due to low sensitivity and the requirement that the macromolecule be small and rapidly tumbling. 7 The requirement for a well-ordered 2D crystal severely limits the use of diffraction techniques to study membrane proteins in native environments. Thus obtaining high resolution in-cell information for membrane proteins remains a challenge that necessitates new approaches.…”

mentioning

confidence: 99%

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

2016

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An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron-electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and the fact that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification and reconstitution usually required for these systems. With this approach structure, function, conformational changes and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment.

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confidence: 99%

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

2016

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“…Like NMR, pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER) is a tool with the potential to examine conformational changes in biomolecules in the cellular environment. [2] PELDOR enables distance measurements in the 1.5–8 nm range between two paramagnetic centers with high precision and reliability. [3, 4] It is more sensitive than NMR and there is no size limit to the protein of interest, both important features for in-cell spectroscopy.…”

mentioning

confidence: 99%

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy

Joseph¹,

Sikora²,

Bordignon³

et al. 2015

Angew Chem Int Ed

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Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin labeled in whole cells and outer membranes and interspin distances were measured to a spin labeled cobalamin using pulse EPR. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.

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“…The PRE-based techniques have been utilized to determine the conformations of ganglioside GM1 embedded in phospholipid bicelles [74] and for characterizing the interaction of amyloid β with GM1 clusters [75]. Paramagnetic probes are currently developed for magnetic resonance analysis of biomolecules in cellular environments [76,77]. Design and creation of novel paramagnetic probes will open up new avenues for in-cell and on-cell NMR approaches, as well as magnetic resonance imaging toward the visualization of dynamic biomolecular processes of glycobiological interest.…”

Section: Perspectivesmentioning

confidence: 99%

Paramagnetic NMR probes for characterization of the dynamic conformations and interactions of oligosaccharides

Kato

Yamaguchi

2015

Glycoconj J

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Paramagnetism-assisted nuclear magnetic resonance (NMR) techniques have recently been applied to a wide variety of biomolecular systems, using sophisticated immobilization methods to attach paramagnetic probes, such as spin labels and lanthanide-chelating groups, at specific sites of the target biomolecules. This is also true in the field of carbohydrate NMR spectroscopy. NMR analysis of oligosaccharides is often precluded by peak overlap resulting from the lack of variability of local chemical structures, by the insufficiency of conformational restraints from nuclear Overhauser effect (NOE) data due to low proton density, and moreover, by the inherently flexible nature of carbohydrate chains. Paramagnetic probes attached to the reducing ends of oligosaccharides cause paramagnetic relaxation enhancements (PREs) and/or pseudocontact shifts (PCSs) resolve the peak overlap problem. These spectral perturbations can be sources of long-range atomic distance information, which complements the local conformational information derived from J couplings and NOEs. Furthermore, paramagnetic NMR approaches, in conjunction with computational methods, have opened up possibilities for the description of dynamic conformational ensembles of oligosaccharides in solution. Several applications of paramagnetic NMR techniques are presented to demonstrate their utility for characterizing the conformational dynamics of oligosaccharides and for probing the carbohydrate-recognition modes of proteins. These techniques can be applied to the characterization of transient, non-stoichiometric interactions and will contribute to the visualization of dynamic biomolecular processes involving sugar chains.

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In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

Cited by 94 publications

References 118 publications

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy

Paramagnetic NMR probes for characterization of the dynamic conformations and interactions of oligosaccharides

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