The Use of 13 C Direct-Detect NMR to Characterize Flexible and Disordered Proteins

• Published in: Methods in enzymology Vol. 611; p. 81
• Main Authors: Cook, Erik C, Usher, Grace A, Showalter, Scott A
• Format: Journal Article
• Language: English
• Published: United States 2018
• Subjects: Amino Acid Sequence; Animals; Carbon Isotopes - analysis; Homeodomain Proteins - chemistry; Humans; Intrinsically Disordered Proteins - chemistry; Nitrogen Isotopes - analysis; Nuclear Magnetic Resonance, Biomolecular - methods; Protein Conformation; Trans-Activators - chemistry; Posttranslational modification; Intrinsically disordered protein; Nuclear magnetic resonance; Chemical shift assignment

NMR spectroscopy remains the only experimental technique that provides (near) atomistic structural information for intrinsically disordered proteins (IDPs), but their sequence and structure characteristics still pose major challenges for high-resolution spectroscopy. Carbon-13 direct-detect NMR spectroscopy can overcome poor spectral dispersion and other difficulties associated with traditional H-detected NMR of nonaggregating disordered proteins. This chapter presents spectroscopic protocols suitable for complete characterization of IDPs that rely exclusively on C direct-detect experiments. The protocols described span initial characterization and chemical shift assignment; structure constraint through residual dipolar coupling and paramagnetic relaxation enhancement measurements; and assessment of intramolecular dynamics through N spin relaxation. The experiments described empower investigators to establish molecular mechanisms and structure-function relationships for IDPs and other proteins characterized by high internal flexibility.