Interaction of β-Hairpin Peptides with Membranes

Project Summary/Abstract
β-Barrels proteins exist in the outer membranes of Gram-negative bacteria, mitochondria, and
chloroplasts. These proteins are composed of a sequence of covalently connected β-hairpins.
Here, we propose to study the interactions of β-hairpin peptides with membranes, namely,
binding, folding, insertion, and self-association. In this process, we will develop a foundation to
understand the early steps of the interactions of β-barrel proteins in membranes, leading to the
test of formation of multimeric barrels from β-hairpins. In the long-term we seek to develop
methods for the design and folding of modular β-barrel proteins in membranes. The model we
have in mind is a small membrane β-barrel protein, which consists of four β-hairpins in one
polypeptide chain (eight strands).
In this project specifically, we propose to design shorter peptides, of about 20-30 residues, that
form β-hairpins, and to study their incorporation in synthetic lipid membranes. We also plan to
understand the rules that determine proper membrane insertion and assembly of these
peptides. The specific aims are to study the thermodynamics and kinetics of binding of β-
hairpins to membranes, their structure in water and in the membrane, their insertion and
orientation, pore formation, and the effect of lipid composition. Critical to these objectives is to
understand how the peptide sequence determines binding, insertion, and folding. These
studies are best performed in simple and easily controlled model systems, using biophysical
methods with designed peptides and model lipid bilayer membranes.
The study of folding of β-barrel membrane proteins, like that of membrane helical proteins, is
complicated mainly because of the unfolded state of the protein in solution, where the nonpolar
(hydrophobic) residues are exposed to water. Therefore, these proteins aggregate when
unfolded in water. One approach to keep the protein monomeric in aqueous solution is to use
detergents or denaturants. Instead, the idea here is to use small units—hairpins—that remain
soluble because they are small. This allows us to study the entire process, from binding from
aqueous solution to insertion, under equilibrium conditions, without co-solvent additives.
A designed modular β-barrel would open the opportunities for applications enormously. The
long-term idea is to engineer binding sites for small molecules, such as drugs, metabolites, or
metal ions, thereby adding and a function to these modular β-barrels, with a large health-
related potential set of applications. The modular nature will also allow the design of
heteromultimeric β-barrels, by combination of different hairpins. Surmounting cellular
membrane barriers, including intracellular compartments, is a major difficulty in the use of
antibiotics, in drug delivery, and in molecular therapy. This proposal addresses the initial steps
to the mechanisms required for a successful approach.

Grant Number: 1R15GM146166-01A1
NIH Institute/Center: NIH
Principal Investigator: PAULO ALMEIDA