脂肪酸結合蛋白(FABPs)為一群受質廣泛、結構同源的動物性小形護送蛋白質。除哺乳類動物細胞中具組織特異性外，腦型脂肪酸結合蛋白近期於模式生物的細胞、及動物行為研究中，被發現與記憶固化相關。過去數十年，獲取較高構形同質性之脂肪酸結合蛋白、以進行結構為主之研究的優化程序一直存有困擾；因此，瞭解脂肪酸結合蛋白於生物膜之間的作用、及運輸受質的方向之細部機制，仍具挑戰性。於此，吾人使用基因重組之果蠅腦型脂肪酸結合蛋白 B 型異構(dFABP)作為模型蛋白質，以研究尿素(urea)純化中併入、或無併入低濃度鹽酸胍(GuHCl)，其同質構形-異質構形之平衡狀態。實驗發現，重複使用鹽酸胍的步驟，可成功獲得高構形同質之 dFABP，以利進一步分析以結構為主的功能性研究。為得此結論，吾人將遠紫外光圓形光譜儀、與內源性螢光所測之熱穩定性作比較；於受質滴定實驗中，氮同位素標記之異核單量子相干磁共振光譜進一步支持，經鹽酸胍純化所得之 dFABP，其構形變化與生化檢驗變化一致。此外，比較晶體結構而得之位置型水分子，顯示鹽酸胍純化所得的蛋白質結構，有較接近空的蛋白質型態，而使以結構為基礎的功能性研究更接近真實。較高的構形同質性可藉由生物膜模擬物–磷脂奈米盤(nanodisc)，揭露脂肪酸、果蠅脂肪酸結合蛋白、與膜間之交互作用，窺知 dFABP 將受質由膜上攜出、並自膜解離，以進行核膜轉運的可能機制。此一關鍵資訊能支持未來多種脂肪酸結合蛋白之研究，例如：FABP 的主動運輸、以將脂肪酸結合蛋白轉入細胞核，及 FABP 與細胞核內接受器作用、以調控基因。

Fatty acid-binding proteins (FABPs) belong to a group of small escort proteins sharing structural homology with broad binding specificity in animal. In addition to their tissue specificity in mammalian cells, brain-type FABP in model animal have been investigated to be related to memory consolidation in cellular and animal behavior studies currently. It has been puzzled with optimization procedures of higher conformational homogeneous FABPs for structure-based study over decades. Thus, the detailed mechanism for ligand transport and its direction among membranes escorted by FABPs remains challenging. Herein, we employed recombinant Drosophila brain-type FABP isoform B (dFABP) as a model protein to study its homogeneous-heterogeneous equilibrium in the presence or absence of low concentration of guanidine hydrochloride (GuHCl) in combination with urea unfolding purification procedure. We found the repeated treatment with GuHCl allowed dFABP to be successfully purified with higher homogeneity that would enable analyses of structure-based functionality. To draw this conclusion, thermal stabilities were compared by far-UV circular dichroism (CD) and intrinsic fluorescence. The 1H-15N heteronuclear single quantum coherence spectra (HSQC) upon ligand titration is further supported the conclusion that the structural change is consistent with biochemical assay in GuHCl-purified dFABP. Besides, the comparison of positional water molecules from crystal structures claims that the structure of GuHCl purification possesses substantial apo-dFABP, which would be more authentic for structural based unctional research. With unraveling the relationship of interaction among ligand-dFABP- membrane by membrane mimetic lipid nanodiscs, higher conformational homogeneity gave a glimpse to the mechanism that ligand may be transferred from membrane by dFABP and dissociated together for further nucleo-cytoplasmic trafficking. This critical information will subsequently support copious FABP studies such as active transport for FABP nuclear translocation and interaction of FABP with nuclear receptors for gene regulation.

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