Designing and engineering polypeptides that have specific structures or functions in biological membranes remains one of the more intractable problems in bioengineering. Although the general principles of binding, folding and self-assembly in membranes are well known1,2
, the details are not understood well enough for rational de novo
design. Furthermore, examples of membrane protein structures are also relatively uncommon, and the known examples lack the high diversity of structural classes that would be useful for homology-based, or templated design.
One desirable bioengineering objective that could lead to new biosensors, antibiotics and cell penetrating peptides is the design of polypeptides that destabilize the permeability barrier of lipid bilayer membranes. For this reason, a great deal of effort has been put into trying to understand the structure-function relationships of known membrane-destabilizing peptides, and some attempts at rational design have been reported e.g.3-5
. Despite a vast literature, compelling structure-function relationships in this field are very rare. Instead, recent literature suggests strongly that some important functions of peptides in biological membranes, such as pore formation6-8
, antimicrobial activity6,9-12
or membrane translocation of some peptides and attached cargo molecules13-16
are not dependent on specific amino acid sequences or three-dimensional peptide structures. Instead they depend on interfacial activity, which is defined here as the ability of a molecule to partition into in the membrane-water interface and to alter or strain the packing and organization of the lipids. Interfacial activity depends mainly on the appropriate balance of physical-chemical interactions between and among peptides, water and membrane lipids, which depend more on the amino acid composition of a peptide than on its exact sequence17
The hundreds of known membrane-active, antimicrobial peptides (AMP) provide many good examples in which specific sequences or three-dimensional structures are apparently not required for their biological activity6,9-12
. Growing evidence suggests that some cell penetrating peptides may function by a similarly non-specific mechanism18
. The activity of such molecules in vitro
and in vivo
depends on their propensity to bind to membranes and self-assemble into peptide-lipid domains that alter membrane permeability. For antimicrobial peptides, this hypothesis explains why compelling structure-activity relationships are so difficult to find. For example, Hancock and colleagues10
made 50 random scrambled sequence variants of a potent 12-residue, membrane-permeabilizing, antimicrobial peptide named Bac2A. They found that about 50% of the scrambled sequences had antimicrobial activity that was at least as good as the parent compound, and 20% of the scrambled sequences had activity that was better than the parent peptide. Upon further analysis these authors concluded, “Sequence alignments of the scrambled Bac2A peptides failed to demonstrate any correlation between peptide sequence and activity”10
. Subsequent QSAR analysis revealed several broad features that correlate with activity, including having a “hydrophobic patch” anywhere in the molecule and having a certain distance between cationic residues. These vague descriptors relate mostly to global hydrophobicity, and thus are consistent with the idea that physical chemical and interfacial properties are the critical factors for determining the biological activity of membrane-destabilizing peptides. Other studies in the literature, including our own work, strongly support this idea3,6,8,12,19-22
. Based on this alternative view of peptide function in membranes we hypothesize that structure-based rational design of membrane active peptides will not be effective, and that potent pore forming peptides might be more effectively selected from libraries that vary a peptides composition
instead of peptides or libraries designed with a particular structure
in mind. In this work, we test the hypothesis and find results that support it.
The interfacial activity of membrane-destabilizing peptides is recapitulated in synthetic bilayer vesicles7,23,24
, which can thus serve as model systems for membrane permeabilizing and membrane translocating peptides. We have previously used such model systems in a structure-based approach to find pore forming peptides7,9
. Here we take a non-classical approach to biomolecular engineering and design of membrane-active peptides by using libraries with rationally designed composition
coupled with a function-based screening method. We have designed compositionally constrained, combinatorial peptide libraries to select peptides that are small and soluble, but which also bind to membranes and induce membrane permeability at low peptide concentration. We describe here the characteristics of the potent pore formers selected from this library and detail their mechanism of action in synthetic phospholipid bilayers. Although they share little sequence similarity in variable positions, the most active molecules share physical properties such as hydrophobicity, and they share the same secondary structure and mechanism of action. The observed mechanism is entirely consistent with having its basis in non sequence-specific interfacial activity. This information leads us to propose a new general model for the mechanism of action of these peptides in membranes. We show here that composition-space peptide libraries coupled with function-based high-throughput screens can lead to the discovery of diverse, soluble and potently membrane-active peptides.