Nuclear magnetic resonance spectroscopy studies of the lipid-bound, receptor active conformation of the apolipoprotein E amino-terminus

Apolipoprotein (apo) E is an exchangeable apolipoprotein that is critical for the trafficking of lipid and cholesterol nutrients in the brain and peripheral circulation. ApoE is a 299 amino acid (37 kDa) protein comprised of two independently folded functional domains, the carboxy-terminal lipid-binding domain and the receptor-binding amino-terminal (NT) domain that only displays receptor competent activity upon association with lipid. In the absence of lipid, the isolated NT domain (residues 1-183) of apoE adopts an amphipathic four alpha-helix bundle architecture that is characteristic of several other related apolipoproteins.;Various models have been advanced that describe the predicted conformational change of the protein upon lipid binding. Experiments have shown that the alpha-helical secondary structure is preserved if not enhanced upon lipid binding and yet it is known that the protein undergoes a dramatic conformational change in the transition to the lipid-bound state. Low-resolution experiments have provided insight into the mechanism and possible path of this transition, but a high-resolution determination of the lipid-bound conformation of apoE has not been accomplished. Using a combination of unique protein engineering methods and nuclear magnetic resonance (NMR) spectroscopy, this thesis advances the understanding of the lipid-induced conformational change of the apoE N-terminus.;Recombinant apoE NT (residues 1-183) is a representative model for apolipoprotein helix bundle conformational flexibility in the presence of lipid and on the surface of lipoprotein particles. The 22 kDa domain is predominantly alpha-helical, monomeric, and comparably stable relative to the native protein. This domain readily forms discoidal particles in the presence of phospholipids, which imparts low-density lipoprotein (LDL) receptor activity to the protein.;A protein engineering approach was used to further define the structural determinants of apoE NT that are necessary for lipid binding. A short helix connecting helix 1 and 2 in the four-helix bundle was replaced by a sequence predicted to adopt a beta-turn. The resulting stable recombinant protein was not compromised in its ability to function as a ligand for the LDL receptor, yet the protein displayed greatly enhanced binding affinity for lipid as assessed by phospholipid solubilization studies, a lipophilic fluorescent dye binding assay, and protection against phospholipase induced aggregation of human LDL fractions.;In order to define the detailed lipid-induced conformational change in apoE, a protein engineering approach termed segmental isotope labeling was deemed necessary to simplify the system for analysis by NMR. Using expressed protein ligation (EPL) methodology, a hybrid apolipoprotein was constructed from two independently generated fragments, apoE residues 1-111 and a 91 amino acid apolipophorin protein fragment. This protein ligation experiment tested the novel use of a pelB leader sequence for the generation of an N-terminal cysteine-containing protein fragment required for the joining of protein fragments by EPL.;Expressed protein ligation techniques were alternatively adapted to create an intact, semisynthetic apoE NT domain using apoE(1-111) and apoE(112-183) protein fragments. This semisynthetic protein displayed nearly identical structural and function properties as wild-type apoE NT by circular dichroism spectroscopy, guanidine denaturation studies, and functional lipid and LDL receptor binding studies. Stable isotope-labeled (15N) apoE(112-183) was produced and ligated to unlabeled apoE 1-111 protein to create a segmental isotope-labeled protein. NMR experiments of the segmental protein further confirmed a structural and functional correspondence between wild-type, fully 15N-labeled and segmental isotope-labeled apoE NT while affirming that the segmental system dramatically simplified the NMR system for examining the protein region containing the LDL receptor recognition sequence.