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Daniilidis, M.* ; Sperl, L.E.* ; Müller, B.S.* ; Babl, A.* ; Hagn, F.

Efficient segmental isotope labeling of integral membrane proteins for high-resolution NMR studies.

J. Am. Chem. Soc. 146, 15403-15410 (2024)
DOI PMC
Creative Commons Lizenzvertrag
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Multidomain Proteins; Structural-analysis; Coupled Receptor; Split Intein; Ligation; Assemblies; Modulation; Assignment; Nanodiscs; Dynamics
ISSN (print) / ISBN 0002-7863
e-ISSN 1520-5126
Zeitschrift Journal of the American Chemical Society
Quellenangaben Band: 146, Heft: 22, Seiten: 15403-15410 Artikelnummer: , Supplement: ,
Verlag American Chemical Society (ACS)
Verlagsort 1155 16th St, Nw, Washington, Dc 20036 Usa
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Structural Biology (STB)
Förderungen Technical University of Munich and Helmholtz Munich
Deutsche Forschungsgemeinschaft
, Deutsche Forschungsgemeinschaft