Structural studies and functional characterization of exchangeable apolipoproteins in lipoprotein formation and transport

My Ph.D. thesis focuses on structural studies of two important exchangeable apolipoproteins, apoE and apoAI. Apolipoproteins are structural components of lipoproteins, including high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very low-density lipoprotein (VLDL), which play critical roles in lipoprotein transport and metabolism. ApoE and apoAI are critical to several major human diseases, including Atherosclerosis and Alzheimer's disease (AD). My Ph.D. thesis is centered on three major objectives.;Objective 1: Structural and functional characterization of apoAI in HDL formation, maturation and transportation.;I have determined NMR structure of apoAI(173-243) in the presence of DPC micelles. I also carried out functional characterizations of different N-terminal truncation mutants and demonstrated that the helix-bundle structure in the apoAI N-terminal domain dictated the formation of different HDL particles, including discoidal and spherical HDLs. This result, for the first time, suggests a regulation role for the helix-bundle structure of apoAI during HDL formation and maturation, providing structural basis of HDL assembly by apoAI protein.;Further, I have been involved in engineering the lipid free apoAI to a biologically active monomer that is amenable for NMR studies. I also worked on NMR structural studies of mouse apoAI/prebeta-HDL which is a 70 kDa protein-lipid complex. I have achieved a partial NMR spectral assignment (160-residues out of 240-residues) and carried out structural characterization of mouse apoAI/prebeta-HDL based on the chemical the shift data.;Objective 2: Determination of the NMR structure of the complete receptor-binding domain of human apoE.;I have determined high-resolution NMR structure of the complete receptor-binding domain of human apolipoprotein E3 (apoE3-NT). I identified unique structural features in the NMR structure. The segments in the N- and C-terminus, which are missing in the crystal structure, form alpha-helices that have extensive tertiary contacts with the bundle, orienting these short helices at specific positions for receptor-binding activity. Several buried hydrophilic residues are observed in the bundle that form buried H-bonds. These buried hydrophilic residues and buried H-bonds are strategically located in specific helix-helix interfaces that may play key roles in a preferred helix-bundle opening and specific helix-bundle recovery due to reversible lipoprotein-binding activity of apoE. A short helix, nHelix C, is fully solvent exposed and nearly perpendicular to the bundle, and likely plays a critical role in initiating protein-lipid interaction, causing this preferred bundle opening. Thus, the NMR structure suggests a unified scheme for initiation and helix-bundle opening of apoE-NT upon lipoprotein-binding.;Objective 3: Development of novel bacterial expression protocol for production of 15-35 milligrams of purified recombinant protein from a 50 milliliter of bacterial cell culture.;Two high cell-density bacterial expression methods have been explored, including an auto-induction introduced by Studier recently and a high cell-density IPTG-induction method described in this study, to achieve a cell-density OD 600 of 10-20 in the normal laboratory setting. Several practical protocols have been implemented with these high cell-density expression methods, to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14-25 milligrams of triple-labeled proteins and 15-35 milligrams of unlabeled proteins from a 50-ml cell culture for all seven different proteins we tested. Such a high protein yield used the same DNA constructs and bacterial strains that we previously used for the traditional IPTG method. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.