title: Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods.
abstract: There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor (1)H amide chemical shift dispersion typically observed in their spectra. Recently, (13)C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multidimensional experiments based on the (15)N,(13)C CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR-based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two three-dimensional (3D) experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy chain connections to be built along the IDP backbone, facilitated by acquisition of amino acid-specific (15)N,(13)C CON-detected experiments. Assignments are completed through carbon-detected, total correlation spectroscopy (TOCSY)-based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through our website.
authors: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
authors: Debashish
authors: D
authors: Sahu
authors: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
authors: Monique
authors: M
authors: Bastidas
authors: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA. Electronic address: sas76@psu.edu.
authors: Scott A
authors: SA
authors: Showalter
mesh_terms: D000595:Amino Acid Sequence; D002247:Carbon Isotopes / Q000032:analysis; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary; D015534:Trans-Activators / Q000032:analysis
chemical_list: D002247:Carbon Isotopes; D018398:Homeodomain Proteins; D064267:Intrinsically Disordered Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein
keywords: Amino acid-filtered NMR; CAS–NMR; Carbon detect NMR; Intrinsically disordered proteins; Pdx1