Fluorescence energy transfer distance measurements using site-directed single cysteine mutants. The membrane insertion of colicin A

The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of insertion of a soluble protein into a lipid bilayer. The soluble structure is known from X-ray crystallography and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this work fluorescence spectroscopy was used to study the membrane-bound structure. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulfol-naphthyl)ethylenediamine (IAEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Three mutants K39C (helix 2), T127C (between helices 6 and 7) and S16Crpt (helix 1, which bears a decapeptide repeat before the mutation) gave useful derivatives. In the soluble protein they showed emission wavelengths decreasing in the order K39C greater than T127C greater than S16Crpt and although all showed blue shifts on addition of dimyristoylphosphatidylglycerol (DMPG) this order was maintained in the membrane-bound state. These shifts were not indicative of deep membrane insertion. Polarization of IAEDANS revealed differences in mobility between mutants. The three tryptophan residues were used as a compound donor to IAEDANS in resonance energy transfer distance determinations. The values obtained for the soluble form were 1.2 A to 3.2 A longer than in the crystal structure. On addition of lipids the indicated distances increased: S16Crpt-I(AEDANS) 6.45 A (22%), K39C-I 5.45 A (18%) and T127C-I 2.4 A (14%). N-bromosuccinimide (NBS) completely abolishes the tryptophan emission from the thermolytic fragment. When lipids were added to a mixture containing ten NBS-treated channel-forming fragments to one IAEDANS labelled fragment the indicated distances increased rather more: S16Crpt-I 9.7 A (38%), K39C-I 8.1 A (36%) and T127C-I 2.5 A (16%). This showed that intermolecular transfer reduces the distance estimated in samples containing only labelled protein. The ensemble of results shows that the amphipathic helices of the C-terminal fragment open out on the surface of the lipid bilayer during the initial phase of membrane insertion.