title: Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study.
abstract: The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Massotte
authors.forename: M
authors.initials: M
authors.lastname: Yamamoto
authors.forename: S
authors.initials: S
authors.lastname: Scianimanico
authors.forename: O
authors.initials: O
authors.lastname: Sorokine
authors.forename: A
authors.initials: A
authors.lastname: van Dorsselaer
authors.forename: Y
authors.initials: Y
authors.lastname: Nakatani
authors.forename: G
authors.initials: G
authors.lastname: Ourisson
authors.forename: F
authors.initials: F
authors.lastname: Pattus
mesh_terms: D002918:Chymotrypsin; D016971:Citrobacter freundii / Q000737:chemistry; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014357:Trypsin
chemical_list: D003087:Colicins; D008055:Lipids; D008565:Membrane Proteins; D002918:Chymotrypsin; D014357:Trypsin