STRUCTURE OF THE MEMBRANE-BOUND FORM OF THE PORE-FORMING DOMAIN OF COLICIN-A - A PARTIAL PROTEOLYSIS AND MASS-SPECTROMETRY STUDY

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertio n of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helic al hairpin. In this study, partial proteolysis and mass spectrometry w ere used to determine the accessible sites to proteolytic attack by tr ypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method f or the identification of the cleavage products, even with partially pu rified peptide mixtures and with only few controls by N-terminal seque ncing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not p rotected by the lipid bilayer from proteolytic degradation. In the abs ence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.