title: Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.
abstract: Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM-MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM-MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI-MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H](+) to [M + 21H](+) and [M + 24H](+) to [M + 29H](+). The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm(2) for the lower charges and 66 ± 6 nm(2) for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53-54 nm(2) following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm(2). These data demonstrate that IM-MS is a valuable tool in studying proteins with different discrete conformational states.
authors: School of Chemistry, University of Nottingham, Nottingham, UK.|School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
authors: Daniel
authors: D
authors: Scott
authors: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
authors: Robert
authors: R
authors: Layfield
authors: School of Chemistry, University of Nottingham, Nottingham, UK.
authors: Neil J
authors: NJ
authors: Oldham
mesh_terms: D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D018583:Pliability; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D007477:Ions; D011994:Recombinant Proteins; D010450:Endopeptidases; C091939:ubiquitin isopeptidase
keywords: Animal proteomics; Electrospray ionization; Ion mobility-mass spectrometry; Protein conformation; Ubiquitin specific protease 5