title: Analysis of Translocation-Competent Secretory Proteins by HDX-MS.
abstract: Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.
authors.affiliation: KU Leuven, Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, Leuven, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Tsirigotaki
authors.affiliation: IMBB-FoRTH, Iraklio, Greece.
authors.forename: M
authors.initials: M
authors.lastname: Papanastasiou
authors.affiliation: University of Southern Denmark, Odense M, Denmark.
authors.forename: M B
authors.initials: MB
authors.lastname: Trelle
authors.affiliation: University of Southern Denmark, Odense M, Denmark.
authors.forename: T J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.affiliation: KU Leuven, Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, Leuven, Belgium; IMBB-FoRTH, Iraklio, Greece. Electronic address: tassos.economou@kuleuven.be.
authors.forename: A
authors.initials: A
authors.lastname: Economou
mesh_terms: D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome; D011994:Recombinant Proteins
keywords: Comparative HDX; Disorder; Exported proteins; Hydrogen/deuterium exchange; Local HDX; Mass spectrometry; Pulsed HDX; Sec secretion; Statistical analysis; Translocation