Interfacial properties of the N-terminal lipid-binding domains of apolipoprotein B and their role in triacylglyceride-rich lipoprotein assembly

ApolipoproteinB (ApoB) is the principal protein component of triacylglyceride (TAG)-rich lipoproteins that are responsible for distribution of hydrophobic lipids to peripheral tissues and are the precursors to low density lipoprotein (LDL). TAG-rich lipoprotein assembly is initiated by the N-terminus of ApoB co-translationally binding and remodeling the luminal leaflet of the rough-ER. In this thesis, the adsorption and interfacial remodeling of the two N-terminal lipid-binding domains of ApoB (ApoB6-13 and B13-17) were characterized at a triolein/water (TO/W) and TO/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/W interface using drop tensiometry. A method was developed to form a monolayer of POPC at a TO/W interface and protocols were developed to characterize the exchangeability, surface pressure (pi)-area isotherm, and exclusion pi (piEX) at a TO/W or TO/POPC/W interface. A model for the behavior of amphipathic alpha-helices at a lipid/water interface was investigated by comparing the N- and C- termini of ApoA-I ([1-44]ApoA-I (N44) and [198-243]ApoA-I (C46)) using these protocols. Both helical peptides had a pi dependent surface conformation and had a higher affinity for a TO/POPC/W interface than a TO/W interface.;The N-terminal lipid-binding domain of ApoB (ApoB6-13), the alpha-helical domain (alphaHD), has 17 alpha-helices with a heterogeneous amphipathic cross-section. The second lipid-binding domain, the C-Sheet (ApoB13-17), is 6 amphipathic beta-strands. The adsorption, stress response, and viscoelasticity of the alphaHD and C-Sheet domains were generally consistent with the behavior of model amphipathic alpha-helices and beta-strands, respectively. The alphaHD domain had a higher affinity for a TO/POPC/W interface than a TO/W interface, while the C-Sheet had a higher affinity for a TO/W interface. POPC shielded the C-Sheet from binding TO. The alphaHD adsorbed to a TO/W interface in a compact conformation where only the N-terminal eight helices interacted with the surface. When the protein monolayer was decompressed, the C-terminal helices bound the lipid. When adsorbed ApoB6-13 was compressed, the N-terminal four helices were progressively expelled from the surface. The remodeling occurred at five distinct transition points where the helices rearranged. There was no evidence of major remodeling of the C-Sheet during expansion and compression. This evidence was used to develop a more detailed model for the initiation of TAG-rich lipoprotein assembly.