title: Time window expansion for HDX analysis of an intrinsically disordered protein.
abstract: Application of typical HDX methods to examine intrinsically disordered proteins (IDP), proteins that are natively unstructured and highly dynamic at physiological pH, is limited because of the rapid exchange of unprotected amide hydrogens with solvent. The exchange rates of these fast exchanging amides are usually faster than the shortest time scale (10 s) employed in typical automated HDX-MS experiments. Considering the functional importance of IDPs and their association with many diseases, it is valuable to develop methods that allow the study of solution dynamics of these proteins as well as the ability to probe the interaction of IDPs with their wide range of binding partners. Here, we report the application of time window expansion to the millisecond range by altering the on-exchange pH of the HDX experiment to study a well-characterized IDP; the activation domain of the nuclear receptor coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). This method enabled mapping the regions of PGC-1α that are stabilized upon binding the ligand binding domain (LBD) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). We further demonstrate the method's applicability to other binding partners of the IDP PGC-1α and pave the way for characterizing many other biologically important ID proteins.
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, 33458, USA.
authors.forename: Devrishi
authors.initials: D
authors.lastname: Goswami
authors.forename: Srikripa
authors.initials: S
authors.lastname: Devarakonda
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Chalmers
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Pascal
authors.forename: Bruce M
authors.initials: BM
authors.lastname: Spiegelman
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D064267:Intrinsically Disordered Proteins; D018160:Receptors, Cytoplasmic and Nuclear