Determining the membrane topology of proteins: Insertion pathway of a transmembrane helix of annexin 12

We describe a sensitive method for determining the bilayer topology of single-site cysteine-linked NBD fluorescent labels on membrane proteins. Based upon a method developed for peptides [W. C. Wimley and S. H. White (2000) Biochemistry 39, 161-170], it utilizes a novel fluorescence quencher, lysoUB, comprised of a single acyl chain attached to a UniBlue chromophore. The enhanced sensitivity of the method arises from the brightness of the NBD fluorescence and the quenching efficiency of lysoUB, which is not fluorescent. In the course of validating the method, we examined the insertion topology of the D-E helical region of repeat 2 of annexin 12, known to adopt a transbilayer orientation at mildly acidic pH [Langen et al. (1998) Proc. Natl. Acad. Sci. USA 95, 14060-14065]. In the final membrane-inserted state, an NBD label attached to the single-cysteine mutant D134C was found to be in the outer (cis) leaflet, while the one attached to D162C was found in the trans leaflet. But kinetic measurements of NBD fluorescence suggested the existence of a transient intermediate insertion state whose lifetime could be increased by increasing the fraction of anionic lipids in the vesicles. Indeed, the lifetime could be increased for times sufficient for the completion of lysoUB-NBD topology measurements. Such measurements revealed that the D-E region adopts an interfacial topology in the intermediate state with both ends on the cis side of the membrane, consistent with the general concept of interface-directed membrane insertion of proteins.