Hydrogen/Deuterium Exchange Kinetics of Apolipophorin-III in Lipid-free and Phospholipid-bound States

Abstract

Attenuated total reflection Fourier transform infra-red spectroscopy was used to probe the kinetics of hy-drogen/deuterium exchange in Manduca sexta apoli-pophorin-III (apoLp-III). ApoLp-III is an exchangeable apolipoprotein that is made up of five elongated am-phipathic-helices in a helical bundle conformation in the monomeric lipid-free form. Upon interaction with phospholipids, it is postulated to undergo a large con-formational change whereby the hydrophobic interior is exposed, facilitating binding to the lipid surfaces. We have used the lipid-free and dimyristoylphosphatidyl-choline-bound apoLp-III to study the dynamically variable domains in the two forms. Three populations of amide protons varying in their hydrogen/deuterium exchange rates were found to exist: slow, intermediate, and fast exchanging, which could correspond to completely buried, partially buried, and solvent-exposed domains on the protein in both the states. In lipid-free apoLp-III, 36, 12, and 52% of the total residues contributed to the slow, intermediate, and fast exchanging populations , respectively. In the dimyristoylphosphatidyl-choline-bound form, the corresponding distribution was 20, 16, and 64%, representing a 12% increase in the number of exposed residues. The results are discussed in terms of increased solvent accessibility due to gross ter-tiary structural reorganization.