Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers

Abstract

The structures of membrane-bound polypeptides are intimately related to their functions and may change dramatically with the lipid environment. Circular dichroism (CD) is a rapid analytical method that requires relatively low amounts of material and no labeling. Conventional CD is routinely used to monitor the secondary structure of peptides and proteins in solution, for example, in the presence of ligands and other binding partners. In the case of membrane-active peptides and transmembrane proteins, these measurements can be applied to, and remain limited to, samples containing detergent micelles or small sonicated lipid vesicles. Such traditional CD analysis reveals only secondary structures. With the help of an oriented circular dichroism (OCD) setup, however, based on the preparation of macroscopically oriented lipid bilayers, it is possible to address the membrane alignment of a peptide in addition to its conformation. This approach has been mostly used for α-helical peptides so far, but other structural elements are conceivable as well. OCD analysis relies on Moffitt's theory, which predicts that the electronic transition dipole moments of the backbone amide bonds in helical polypeptides are polarized either parallel or perpendicular to the helix axis. The interaction of the electric field vector of the circularly polarized light with these transitions results in an OCD spectrum of a membrane-bound α-helical peptide, which exhibits a characteristic line shape and reflects the angle between the helix axis and the bilayer normal. For parallel alignment of a peptide helix with respect to the membrane surface (S-state), the corresponding "fingerprint" CD band around 208 nm will exhibit maximum negative amplitude. If the helix changes its alignment via an obliquely tilted (T-state) to a fully inserted transmembrane orientation (I-state), the ellipticity at 208 nm decreases and the value approaches zero due to the decreased interactions between the field and the transition dipole. Compared to conventional CD, OCD data are not only collected in the biologically relevant environment of a highly hydrated planar lipid bilayer (whose composition can be varied at will), but in addition it provides information about the tilt angle of the polypeptide in the membrane. It is the method of choice for screening numerous different conditions, such as peptide concentration, lipid composition, membrane additives, pH, temperature, and sample hydration. All these factors have been found to affect the peptide alignment in membrane, while having little or no influence on conformation. In many cases, the observed realignment could be related to biological action, such as pore formation by antimicrobial and cell-penetrating peptides, or to binding events of transmembrane segments of integral membrane proteins. Likewise, any lipid-induced conversion from α-helix to β-sheeted conformation is readily picked up by OCD and has been interpreted in terms of protein instability or amyloid-formation.

Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

“…Each spectrum was averaged over 3 repeat scans. The averaged spectra were normalized to molar ellipticity per residue and the results were analysed following the protocols of Huang and Ulrich2829.…”

Circular dichroism spectroscopy of membrane proteins

“…40,42 This has proven to be a very valuable method for examining antimicrobial and other surfactant peptides. 17 4.3.1 Protocols for collecting oriented CD data. Circular flat plates made of Suprasil quartz or calcium fluoride (the latter especially appropriate for SRCD measurements) are suitable substrates for creating oriented membrane samples.…”

Membrane Active Peptides and Their Biophysical Characterization

“…While amphiphilic peptides form random coil structures in solution, they adopt α-helical or β-sheet structures upon binding to membranes [197]. Conventional CD yields information of a peptide in solution and the structural changes in a peptide upon binding [124,198]. The change in the structure upon binding the membrane can be used to estimate the ratio of bound to free peptides but traditional CD does not yield any information about the orientation of the peptide relative to the membrane.…”

“…The change in the structure upon binding the membrane can be used to estimate the ratio of bound to free peptides but traditional CD does not yield any information about the orientation of the peptide relative to the membrane. Thus, this approach is limited to samples in detergent micelles or liposomes or samples containing small sonicated lipid vesicles [112,198,199]. These studies try to mimic the bacterial outer leaflet, or LPS, to determine the structure of peptides in cell-like environments [124,190].…”

“…It is performed on macroscopically oriented samples to address the membrane alignment of a peptide in addition to its conformation. So far, it has been mainly used for the study of α-helical peptides [32,198,201,202]. Extensions of traditional CD spectroscopy such vibrational and electronic CD also exits as powerful tools for the study of membrane active peptides [203].…”