title: Segmental Isotope Labeling of Insoluble Proteins for Solid-State NMR by Protein Trans-Splicing.
abstract: Solid-state NMR spectroscopy (ssNMR) is uniquely suited for atomic-resolution structural investigations of large protein assemblies, which are notoriously difficult to study due to their insoluble and non-crystalline nature. However, assignment ambiguities because of limited resolution and spectral crowding are currently major hurdles that quickly increase with the length of the polypeptide chain. The line widths of ssNMR signals are independent of proteins size, making segmental isotope labeling a powerful approach to overcome this limitation. It allows a scalable reduction of signal overlap, aids the assignment of repetitive amino acid sequences, and can be easily combined with other selective isotope labeling strategies. Here we present a detailed protocol for segmental isotope labeling of insoluble proteins using protein trans-splicing. Our protocol exploits the ability of many insoluble proteins, such as amyloid fibrils, to fold correctly under in vitro conditions. In combination with the robust trans-splicing efficiency of the intein DnaE from Nostoc punctiforme, this allows for high yields of segmentally labeled protein required for ssNMR analysis.
authors.affiliation: Macromolecular Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany.|Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon/UCBL, University of Lyon, 69100, Villeurbanne, France.
authors.forename: Tobias
authors.initials: T
authors.lastname: Schubeis
authors.affiliation: Macromolecular Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany.|Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Straße 10, D-13125, Berlin, Germany.
authors.forename: Madhu
authors.initials: M
authors.lastname: Nagaraj
authors.affiliation: Macromolecular Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany. christiane.ritter@senostic.com.|SeNostic GmbH, Feodor-Lynen-Strasse 21, 30625, Hannover, Germany. christiane.ritter@senostic.com.
authors.forename: Christiane
authors.initials: C
authors.lastname: Ritter
mesh_terms: D047668:Inteins; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins
keywords: Amyloid; Intein; Isotopic labeling; Protein complexes; Protein structures; Protein trans-splicing; Solid-state NMR spectroscopy