Structural Characterization of Human Apolipoprotein E 72-166 Fragments

• Main Authors: Yi-Hui Hsieh, 謝議輝
• Other Authors: Gu-Gang Chang
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/34402726820699262525

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 95 === 　Human apolipoprotein E (apoE) is composed of 299 amino acids having a molecular weight of 34 kDa. apoE plays important roles in lipid metabolism involving lipid transportation and lipoprotein catabolism. There are three major apoE isoforms: apoE2, apoE3, and apoE4. These three isoforms differ from one another only by amino acids 112th and 158th. We have expressed N- and C-terminal truncated apoE-(72-166) fragments tailored to eliminate helix- and domain interactions to unveil structural and functional disturbances. The secondary structure of three apoE-(72-166) isoforms was stabilized in lipid environment by GdnHCl denaturation experiment. apoE4-(72-166) had lowest secondary structure stability both in aqueous and lipid environment. ANS titration experiment also indicated that apoE4-(72-166) maintained the lowest Kd and highest maximal ANS binding capacity in PBS with or without DHPC, suggesting that apoE4-(72-166) has maximum hydrophobic exposure among three isoforms. apoE4-(72-166) also has lowest tertiary structure stability in GdnHCl denaturation study by fluorescence spectrophotometry. In quaternary structure analysis by analytical ultracentrifugation, three apoE-(72-166) isoforms had complexed oligomerization in aqueous solution, and the quaternary structure could be simplified by the adding of DHPC. The rapid dissociation of quaternary structure of apoE4-(72-166) could be found both in velocity and equilibrium experiments. In the functional assays, apoE-(72-166) still remain beta-VLDL binding ability in vitro. The structural preference of truncated apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiological functions. The apoE4-(72-166) may be a plausible target for the design of a peptide drug to alleviate the propensity to aggregation.