果蠅脂肪酸結合蛋白的結構與功能分析

Structural and Functional Analysis of Drosophila Fatty Acid-Binding Protein

林欣慧(Hsin-Hui Lin)

指導教授 ： 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00580

摘要

脂肪酸結合蛋白質家族可以結合並且運輸疏水性的脂質,而果蠅的脂肪酸結合蛋白(dFABP)即屬於這個家族的一員。dFABP和哺乳類腦型的脂肪酸結合蛋白(FABP7)有很高的序列同源性，且已經被研究出會參與果蠅長期記憶的形成。 然而，以 dFABP作為介導的脂質結合特異性以及長期記憶形成的細胞機制流程都還尚未清楚。這篇研究中，我們想要了解dFABP的結構特性和分析其和脂肪結合的特異性。在圓二色和一維核磁共振光譜中，顯示出dFABP是以β-strand為主要構型的二級結構且是一個以摺疊完整的水溶液結構。另外，圓二色光譜也證明了其結構對於pH質的變化相當敏感。根據ANS的競爭實驗，dFABP2對於長鏈不飽和脂肪酸相較於飽和脂肪酸有很明顯結合偏好的趨勢。而在dFabp/DHA和dFabp/OA的模型建構以及點突變的實驗呈現出Tyr126和Arg124在脂肪結合上扮演著重要的角色。最後根據同源序列的比對和模型表面電荷的分析，我們把dFABP分類在第四個亞族(進行結合時可將脂質摺疊成U字型或高度的折疊)，並歸類為可利用直接碰撞的機制將脂質傳送到膜上。在這些實驗之後，我們得到更多dFABP的結構特性以及對於脂質結合的特異性的資訊，能有助於將來對於形成長期記憶研究的發展。

關鍵字

果蠅脂肪酸結合蛋白

並列摘要

Drosophila fatty acid binding protein isoform B (dFABP) is a member of the fatty acid-binding protein family which can bind and transport hydrophobic ligand. dFABP has the high sequence similarity to mammalian brain-type FABP (FABP7) and has been reported to be involved in the enhancement of long-term memory (LTM) consolidation in Drosophila melanogaster. However, the lipid binding specificities or the cellular mechanism processes of LTM consolidation mediated by dFABP are still unclear. In this study, we attempt to characterize its structural properties and to investigate the biological functions of dFabp. The circular dichroism (CD) and one-dimensional nuclear magnetic resonance (NMR) implied that dFabp shows secondary structure of major β-strand conformation and forms a well-packed solution structure. CD spectra also demonstrate that the stability of structure is sensitive to pH value. According to ANS competitive experiment, dFabp has a dominant binding preference for unsaturated than saturated fatty acids. Furthermore, the structural modeling of dFabp/DHA and dFabp/OA complexes analysis and site-directed mutagenesis experiments both revealed that Tyr126 and Arg124 play important roles in ligand binding. Based on the homology sequence alignments and the surface electric analysis of model, we categorize dFABP into group iv which can fold fatty acid in U-shaped conformation or in a highly bent, and also into the
3
collision-mediated mechanism of lipid transfer. After all, we gain more insights into the fatty acid binding affinities and the structural properties of dFABP for future studies of long-term memory consolidation.

並列關鍵字

dFABP ； Drosophila Fatty Acid-Binding Protein

參考文獻

Reference list

Alpers, D.H., A.W. Strauss, R.K. Ockner, N.M. Bass, & J.I. Gordon. (1984). "Cloning of a cDNA encoding rat intestinal fatty acid binding protein." Proc. Natl. Acad. Sci. USA81: 313-317.

Banaszak, L., N. Winter, Z. H. Xu, D.A. Bernlohr, S. Cowan, &T.A. Jones. (1994). "Lipid-binding proteins- a family of fatty-acid and retinoid transport proteins." Adv. Protein Chem. 45: 89–151.

Gerstner, J.R., J.C. Yin. (2010)."Circadian rhythms and memory formation." Nat Rev Neurosci. 11: 577-88.

Gerstner, J.R., Q.Z. Bremer, W.M. V. Heyden, T.M. LaVaute, J.C. Yin, & C.F. Landry. (2008). "Brain fatty acid binding protein (Fabp7) is diurnally regulated in astrocytes and hippocampal granule cell precursors in adult rodent brain." PLoS ONE. 3(2): e1631.