title: Methods in the Study of PTEN Structure: X-Ray Crystallography and Hydrogen Deuterium Exchange Mass Spectrometry.
abstract: Despite its small size and deceptively simple domain organization, PTEN remains a challenging structural target due to its N- and C-terminal intrinsically disordered segments, and the conformational heterogeneity caused by phosphorylation of its C terminus. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS), it is possible to probe the conformational dynamics of the disordered termini, and also to determine how PTEN binds to lipid membranes. Here, we describe how to purify recombinant, homogenously dephosphorylated PTEN from a eukaryotic system for subsequent investigation with HDX-MS or crystallography.
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council (MRC), Francis Crick Avenue, Cambridge Biomedical campus, Cambridge, CB2 0QH, UK.
authors.forename: Glenn R
authors.initials: GR
authors.lastname: Masson
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Drive, Victoria, BC, Canada, V8P 5C2.
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council (MRC), Francis Crick Avenue, Cambridge Biomedical campus, Cambridge, CB2 0QH, UK. rlw@mrc-lmb.cam.ac.uk.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray / Q000379:methods*; D041961:Deuterium Exchange Measurement / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051059:PTEN Phosphohydrolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D051059:PTEN Phosphohydrolase
keywords: HDX-MS; Insect cell expression; Liposome preparation; Mass spectrometry; Protein purification