Bilayer-Dependent Recognition of Docosahexaenoic Acid by the Transmembrane Domain of FATP3.

IF 2.9 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Membrane Biology Pub Date : 2025-09-15 DOI:10.1007/s00232-025-00361-4

Yi Ding, Yonghua Wang, Wen Chen

引用次数: 0

Abstract

Fatty acid Transport Protein 3 (FATP3) is a single-pass transmembrane protein implicated in the uptake and intracellular transport of long-chain fatty acids, yet the molecular contribution of its transmembrane domain (TMD) remains poorly defined. Here, we establish an efficient and reproducible strategy for heterologous expression, purification, and in vitro reconstitution of FATP3-TMD. FATP3-TMD was over-expressed in Escherichia coli as a TrpLE fusion, liberated by cyanogen-bromide cleavage and polished by one-step reverse-phase HPLC, yielding milligram quantities of highly pure peptide. ¹H-¹⁵N HSQC spectroscopy revealed a well-folded FATP3-TMD in both Fos-choline-14 micelles and DMPC/DHPC bicelles. Strikingly, titration with docosahexaenoic acid (DHA) induced residue-specific chemical-shift perturbations exclusively in bicelles. These data demonstrate that a bilayer-like lipid context is essential for functional recognition of ω-3 fatty acids by the FATP3-TMD and provide a robust platform for mechanistic dissection of FATP3 mediated lipid transport.

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FATP3跨膜结构域对二十二碳六烯酸的双层依赖性识别。

脂肪酸转运蛋白3 （FATP3）是一种单遍跨膜蛋白，参与长链脂肪酸的摄取和细胞内转运，但其跨膜结构域（TMD）的分子作用仍不清楚。在这里，我们建立了一种高效和可重复的策略，用于FATP3-TMD的异种表达、纯化和体外重组。FATP3-TMD在大肠杆菌中以TrpLE融合过表达，通过氰溴裂解释放，并通过一步反相高效液相色谱抛光，产生毫克量的高纯度肽。1H-15N HSQC光谱显示，在fos -胆碱-14胶束和DMPC/DHPC胶束中都有一个折叠良好的FATP3-TMD。引人注目的是，用二十二碳六烯酸（DHA）滴定引起的残基特异性化学位移扰动仅在单胞体中。这些数据表明，双层样脂质背景对于FATP3- tmd对ω-3脂肪酸的功能识别至关重要，并为FATP3介导的脂质转运的机制解剖提供了一个强大的平台。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Membrane Biology 生物-生化与分子生物学

CiteScore

4.80

自引率

4.20%

发文量

审稿时长

6-12 weeks

期刊介绍： The Journal of Membrane Biology is dedicated to publishing high-quality science related to membrane biology, biochemistry and biophysics. In particular, we welcome work that uses modern experimental or computational methods including but not limited to those with microscopy, diffraction, NMR, computer simulations, or biochemistry aimed at membrane associated or membrane embedded proteins or model membrane systems. These methods might be applied to study topics like membrane protein structure and function, membrane mediated or controlled signaling mechanisms, cell-cell communication via gap junctions, the behavior of proteins and lipids based on monolayer or bilayer systems, or genetic and regulatory mechanisms controlling membrane function. Research articles, short communications and reviews are all welcome. We also encourage authors to consider publishing ''negative'' results where experiments or simulations were well performed, but resulted in unusual or unexpected outcomes without obvious explanations. While we welcome connections to clinical studies, submissions that are primarily clinical in nature or that fail to make connections to the basic science issues of membrane structure, chemistry and function, are not appropriate for the journal. In a similar way, studies that are primarily descriptive and narratives of assays in a clinical or population study are best published in other journals. If you are not certain, it is entirely appropriate to write to us to inquire if your study is a good fit for the journal.