A functional protein pore with a "retro" transmembrane domain

Extended retro (reversed) peptide sequences have not previously been accomm odated within functional proteins. Here, we show that the entire transmembr ane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residu es, 25 contributed by the central sequence of each subunit of the heptameri c pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less t han that of the wild-type heptamer and its current-voltage relationship den otes close to ohmic behavior, while the wild-type pore is weakly rectifying . Both wild-type and retro pores are very weakly anion selective. These res ults and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-b arrel, explaining, for example, the activity of the retro forms of many mem brane-permeabilizing peptides. By contrast with the wild-type subunits, mon omeric retro subunits undergo premature assembly in the absence of membrane s, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this dif ficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfav orable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the p resence of subconductance states in single-channel recordings. Knowledge ab out the extraordinary plasticity of the transmembrane beta-barrel of alpha- hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.