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.

More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes

“…Indeed, the ability of such short helices to form a channel is mainly related to their effect on membrane thickness and the induction of curvature in the membrane associated with toroidal pore formation (further comments on this topic in ''Lipids and Proteins Acting Together: The Protein-Lipid Pore'' section) (Parker et al 1990). Alternatively, the ''penknife'' model proposes that the hydrophobic hairpin is only shallowly inserted while laying more or less parallel to the membrane plane (Massotte et al 1993). Another possibility is that the hydrophobic hairpin is in fact alternating between these two different conformations (Kienker et al 1997).…”

Adventures in Membrane Protein Topology

“…The em max data for the contingent of single Trp mutant and WT proteins strongly suggest that the polarity of the Trp residues (at 11 of the 14 different Trp sites within the peptide) increases when the protein associates with (51); biotinylation (41,42,53); epitope mapping (43); saturation mutagenesis (55); protease accessibility (28,47); depth-dependent fluorescence quenching (29,31); cysteine-scanning mutagenesis (56); FRET (27); CD, Fourier transform infrared radiation, and differential scanning calorimetry (48,49); time-resolved spin labeling (57); and acrylamide quenching and other fluorescence data presented in this work. The initial surface-bound structure adopts at least two states with state 2 being the most populated.…”

“…A model for colicin A has been proposed wherein most helices open like an "umbrella" on the surface of the membrane (26). An alternate model, called the "penknife model," has been suggested which proposes that helices 8 and 9 only partially penetrate into the membrane bilayer (27,28).…”

Bioactive Peptides used by Bacteria in the Concur-Rence for the Ecological Niche: General Classification and Mode of Action (Overview)

“…A model for colicin A has been proposed wherein most helices open like an "umbrella" on the surface of the membrane (52). An alternate model, called the "penknife model," has been suggested which proposes that helices 8 and 9 only partially penetrate into the membrane bilayer (53,54).…”