Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL‐ALSOFAST Experiment with Delayed Decoupling

Rößler, Philip; Mathieu, Daniel; Gossert, Alvar D.

doi:10.1002/anie.202007715

Cited by 35 publications

(25 citation statements)

References 50 publications

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“…Altogether, 13 C-edited experiments might have been underexploited for in-cell structural biology by NMR. 13 C-labeling is most often more expensive than 15 N-labeling, but the advantages of 2D 1 H– 13 C correlation spectra show promising results, which could be further improved by some recent isotope labeling methods and pulse sequences. , …”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

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

Theillet

2022

Chem. Rev.

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“…NMR studies of the activated state of tβ 1 AR have so far exclusively been carried out using Nb80 or its affinity maturated analogue Nb6B9, instead of the more native-like G protein surrogate mini-G s , due to the limited stability of mini-G s complexes. Using the newly developed XL-ALSOFAST-HMQC [ 51 ] experiment, we were able to obtain high quality spectra of mini-G s complexes in a matter of hours. Therefore, we can compare the effects of mini-G s and Nb80.…”

Section: Resultsmentioning

confidence: 99%

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

et al. 2020

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In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized β1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with β1 adrenergic receptor (β1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tβ1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.

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“…In the first works describing protein in-cell NMR in E. coli , conventional 15 N and 13 C HSQC spectra were recorded, which were the state-of-the-art at the time. , Since then, several variations of these NMR spectra have been developed, including the so-called fast-pulsing NMR methods, which manipulate differently the magnetization of the observed and nonobserved nuclei in order to greatly reduce the longitudinal relaxation time of the observed nuclei and therefore allow shorter interscan delays with minor signal loss, greatly improving the sensitivity. Among these, the SOFAST-HMQC and BEST-type experiments have found widespread use, , and more recently ALSOFAST-type experiments have further improved the performance of 1 H– 13 C methyl correlation spectra. , For 1 H– 15 N correlation spectra, the SOFAST-HMQC pulse sequence is likely the most commonly used, due to its simplicity and high sensitivity in various sample conditions for both folded , and unfolded proteins and for nucleic acids . Compared to folded proteins, intrinsically disordered proteins have a much lower amide 1 H chemical shift dispersion, which may pose additional challenges for an in-cell analysis.…”

Section: Methodological Aspectsmentioning

confidence: 99%

Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR

2022

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A detailed knowledge of the complex processes that make cells and organisms alive is fundamental in order to understand diseases and to develop novel drugs and therapeutic treatments. To this aim, biological macromolecules should ideally be characterized at atomic resolution directly within the cellular environment. Among the existing structural techniques, solution NMR stands out as the only one able to investigate at high resolution the structure and dynamic behavior of macromolecules directly in living cells. With the advent of more sensitive NMR hardware and new biotechnological tools, modern in-cell NMR approaches have been established since the early 2000s. At the coming of age of in-cell NMR, we provide a detailed overview of its developments and applications in the 20 years that followed its inception. We review the existing approaches for cell sample preparation and isotopic labeling, the application of in-cell NMR to important biological questions, and the development of NMR bioreactor devices, which greatly increase the lifetime of the cells allowing real-time monitoring of intracellular metabolites and proteins. Finally, we share our thoughts on the future perspectives of the in-cell NMR methodology.

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Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL‐ALSOFAST Experiment with Delayed Decoupling

Cited by 35 publications

References 50 publications

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

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

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR

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