Extended retro (reversed) peptide sequences have
not previously been accommodated within functional proteins.
Here, we show that the entire transmembrane portion of
the β-barrel of the pore-forming protein α-hemolysin
can be formed by retrosequences comprising a total of 175
amino acid residues, 25 contributed by the central sequence
of each subunit of the heptameric 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 than that of the wild-type heptamer and its
current-voltage relationship denotes close to ohmic behavior,
while the wild-type pore is weakly rectifying. Both wild-type
and retro pores are very weakly anion selective. These
results and the examination of molecular models suggest
that β-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
β-barrel, explaining, for example, the activity of
the retro forms of many membrane-permeabilizing peptides.
By contrast with the wild-type subunits, monomeric retro
subunits undergo premature assembly in the absence of membranes,
most likely because the altered central sequence fails
to interact with the remainder of the subunit, thereby
initiating assembly. Despite this difficulty, a technique
was devised for obtaining heteromeric pores containing
both wild-type and retro subunits. Most probably as a consequence
of unfavorable interstrand side-chain interactions, the
heteromeric pores are less stable than either the wild-type
or retro homoheptamers, as judged by the presence of subconductance
states in single-channel recordings. Knowledge about the
extraordinary plasticity of the transmembrane β-barrel
of α-hemolysin will be very useful in the de novo design
of functional membrane proteins based on the β-barrel
motif.
