Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.
The Journal of Membrane Biology – Springer Journals
Published: Aug 13, 2010
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