Basic residues in helical region affect protein stability and ligand binding in Drosophila Fatty Acid Binding Protein
| Autor: | Huang, Yun-Fang, 黃韻芳 |
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| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 Drosophila melanogaster Fatty acid-binding protein (dFABP) comprises ten antiparallel β-strands and two α-helixes which is the typical structural feature of FABP family. dFABP was found to play an important role in long-term memory formation recently. In our previous studies, we have identified the critical residue-Y127 and R125 of binding sites in the β-barrel region. The helical region of FABP is referred to as the ligand entering site. Basic residues in this region are considered to be involved in membrane interaction and also act as nuclear location signal. In order to investigate the role of these basic amino acids, we replaced residues K21A, R30A and K31A with alanine using site-directed mutagenesis. The secondary structures of mutants are similar to wild-type dFABP. However, the melting temperature measured by circular dichorism shows a significant increase for all of three mutants. The enhancement of thermal stability may due to the elimination of repulsive force of positive charges on the helical region. Furthermore, isothermal titration calorimetry was employed to examine the binding ability toward fatty acids. Although the binding constant for oleic acid (OA) are similar, R30A and K31A, both on α-II helix, shows a ten-fold decrease in binding affinity to docosahexaenoic acid (DHA) compared to the wild-type dFABP. Our results indicate that basic residues in α-II region play the important role in modulating entrance for ligand binding. The binding affinity of dFABP is carefully balanced between structural stability and flexibility. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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