title: Mapping unstructured regions and synergistic folding in intrinsically disordered proteins with amide H/D exchange mass spectrometry.
abstract: Mapping the structured and disordered regions and identifying disorder-to-order transitions are essential to understanding intrinsically disordered proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore the feasibility of H/D-MS for mapping disordered and ordered regions in IDPs, we undertook a systematic evaluation of an unstructured protein, a molten globular protein, and the well-folded complex of the two proteins. Most segments of the unstructured protein, ACTR (activator of thyroid and retinoid receptors, NCOA3_HUMAN, residues 1018-1088), exchange at rates consistent with its assignment as an unstructured protein, but there is slight protection in regions that become helical in the ACTR-CBP complex. The molten globular protein, CBP (the nuclear coactivator binding domain of the CREB binding protein, CBP_MOUSE, residues 2059-2117), is moderately protected from exchange, and the protection is nearly uniform across the length of the protein. The uniformity arises because of rapid interconversion between an ensemble of folded conformers and an ensemble of unstructured conformers. Rapid interconversion causes the H/D exchange kinetics to be dominated by exchange by molecules in unstructured conformations. For the folded ACTR-CBP complex, the exchange data provide a qualitatively accurate description of the complex. Our results provide a useful framework to use in the interpretation of H/D-MS data of intrinsically disordered proteins.
authors: Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, United States.
authors: Theodore R
authors: TR
authors: Keppel
authors: Brent A
authors: BA
authors: Howard
authors: David D
authors: DD
authors: Weis
mesh_terms: D000818:Animals; D050882:CREB-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D056921:Nuclear Receptor Coactivator 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding
chemical_list: D006859:Hydrogen; D003903:Deuterium; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3