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

Bilayer-Dependent Recognition of Docosahexaenoic Acid by the Transmembrane Domain of FATP3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Bilayer-Dependent Recognition of Docosahexaenoic Acid by the Transmembrane Domain of FATP3 Yi Ding, Yonghua Wang, Wen Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6860198/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Sep, 2025 Read the published version in The Journal of Membrane Biology → Version 1 posted 10 You are reading this latest preprint version 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. 1 H- 15 N 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. FATP3-TMD DHA Micelles Bicelles NMR Figures Figure 1 Figure 2 Introduction Fatty acid transport protein 3 (FATP3), also known as very long-chain acyl-CoA synthetase 3 (ACSVL3), encoded by the gene SLC27A3 , is a member of the solute carrier family 27 (Hirsch et al. 1998). Sequence analysis of FATP3 reveals two conserved functional motifs: an ATP/AMP binding domain (~100 amino acids) and a FATP/VLACS-specific motif (~50 amino acids) unique to the FATP family (DiRusso et al. 2008). FATP3 plays dual roles in lipid metabolism: (1) as an acyl-CoA synthetase, it catalyzes the activation of long-chain and very long-chain fatty acids into acyl-CoA derivatives in an ATP-dependent manner, and (2) it facilitates fatty acid update by enhancing cellular membrane permeability to fatty acids (Pei et al. 2013). However, the extent to which FATP3 functions as a direct transporter remains a subject of ongoing investigation and debate. FATP3 is a membrane-associated protein that contains hydrophobic transmembrane regions within its primary structure. Due to its high sequence with FATP1 (Gimeno 2007), and based on epitope tag assay of FATP1 (Stahl 2004), FATP3 is hypothesized to adopt a similar membrane topology—featuring an N-terminal transmembrane anchor and a C-terminal domain oriented toward the cytoplasm. In addition to the primary transmembrane helix, FATP3 may possess additional membrane-associating regions that facilitate its localization to the endoplasmic reticulum or other membrane compartments (Stahl 2004). To date, no high-resolution structures of FATP3 have been reported, and its precise structural conformation remains to be experimentally determined. Highly unsaturated ω-3 fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are essential for the development and function of neural and retinal tissues. And the mechanisms underlying their transmembrane transport have garnered significant research interest in recent years (Koletzko et al. 2007; Ochiai et al. 2017; Wood et al. 2021). FATP3 has been proposed as a potential mediator of ω-3 fatty acid transport for its expression in barrier tissues (Hagberg et al. 2013; Kappen et al. 2022). For instance, FATP3 is expressed in the placental trophoblasts and in the capillary endothelium of the blood-brain barrier, suggesting a possible role in the transfer of maternal fatty acids to the fetal brain (Maekawa et al. 2015). Although FATP3 exhibits lower expression levels in the adult brain compared to the brain-specific transporter MFSD2A—which facilitates DHA uptake in the form of lysophosphatidylcholine (Lyso-PLA) (Korbecki et al. 2023) —its expression is markedly higher in the embryonic mouse brain and declines after birth (Maekawa et al. 2015). This developmental expression pattern implies that FATP3 may be involved in the provision of essential fatty acids or in membrane lipid synthesis during critical stages of neurodevelopment. Currently, however, direct functional evidence demonstrating that FATP3 specifically facilitates the uptake of DHA or EPA remains scarce. Indirect studies in brain microvascular endothelial cells show that, fatty acid-binding protein FABP5 binds and delivers DHA intracellularly, while both FATP3 and FATP4 are required for effective transendothelial fatty acid flux (Hagberg et al. 2013). Nontheless, there is no conclusive evidence that FATP3 mediates the transmembrane transport of ω-3 fatty acids with high specificity. Its proposed role in ω-3 fatty acid trafficking is largely inferred from tissue-specific expression patterns and indirect functional correlations. To date, FATP3 has not been examined at the molecular level. Because its extracellular domain consists of only two residues, insufficient for high-affinity binding of free fatty acids. We hypothesized that the transmembrane domain of FATP3 mediates fatty acid recognition. We therefore expressed the FATP3-TMD in Escherichia coli as a TrpLE fusion, a strategy that drove high-level accumulation in inclusion bodies and enabled purification of milligram quantities of highly pure protein (Chen and Cotten 2014; Fu et al. 2019). After cyanogen-bromide cleavage and RP-HPLC purification, FATP3-TMD was reconstituted into membrane-mimetic systems, where it adopted the expected α-helical fold and retained functional integrity. 1 H- 15 N NMR titrations demonstrated specific binding of ω-3 polyunsaturated fatty acids such as DHA to FATP3-TMD, providing a molecular foundation for elucidating how FATP3 facilitates DHA transport across biological membranes. Materials and Methods Detailed materials and methods can be found in the supplemantary documents. Construction of recombinant plasmid The nucleotide sequence encoding residues 1-32 of human FATP3 (UniProt Q8N163: MAALLLLPLLLLLPLLLLKLHLWPQLRWLPAD) was codon-optimized for E. coli expression, Synthesized de novo, and subcloned into the pMM-LR6 vector to generate the recombinant expression plasmid pMM-LR6-FATP3-TMD. Protein expression and isotopic labeling E. coli BL21 (DE3) cells harbouring pMM-LR6-FATP3-TMD were grown in LB medium (50 µg/mL kanamycin) until the OD 600 reached 0.6-0.7. Expression was induced with 0.2 mM IPTG, and the culture was shifted to 20℃ for 16 hours. Cells were collected by centrifugation and stored at -80℃. Uniformly 15 N labeled FATP3-TMD was produced under identical conditions in M9 minimal medium supplemented with 15 NH 4 Cl (1 g/L) as the sole nitrogen source. Protein purification Bacterial pellets were disrupted by sonication. Soluble fractions were removed by centrifugation, and the inclusion-body pellet was retained and solubilized in the guanidine buffer. Insoluble material was removed by a second centrifugation step. The supernatant was initially purified by Ni-NTA resin. Cyanogen bromide (CNBr) was added to the eluate and cleavage was carried out. The reaction mixture was transferred to 2 kDa MWCO dialysis tubing and dialyzed twice against ultrapure water, then lyophilized. The dried material was dissolved in 50 % (v/v) formic acid, then purified by RP-HPLC on a C3 column (Agilent, ZORBAX) using an isopropanol/acetonitrile/water gradient containing 0.1 % trifluoroacetic acid. Fractions containing the target FATP3-TMD were pooled, lyophilised, and stored at -20 ℃. Mass spectrometry The molecular weight of the purified FATP3-TMD was confirmed by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. Reconstitution Micelles: Fos-Choline-14 and Lyophilized FATP3-TMD was dissolved and gently mixed in 6 M guanidine in NMR buffer (25 mM MES, pH 6.7). The solution was transferred to a 2 KDa MWCO dialysis cassette (Thermo Scientific) and dialyzed against detergent-free NMR buffer to allow micelle formation and refolding. The dialysate was concentrated to 300 μl by centrifugation, and supplemented with 20 μl of D 2 O. Bicelles ( q ~0.4): DMPC, DH 6 PC and protein sample were co-dissolved in hexafluoroisopropanol. The solvent was evaporated under gentle nitrogen stream, and the mixture was further dried under vacuum. The residue was rehydrated in 6 M guanidine in NMR buffer, and was dialyzed as above to remove denaturant and allow bicelle assembly. After dialysis, the sample was clarified, concentrated to 300 µl, supplemented with 20 µl of D 2 O, and transferred to a Shigemi tube for NMR measurements. NMR titration experiment 15 N- 1 H heteronuclear single-quantum coherence (HSQC) spectra were recorded at 35 ℃ on Bruker Avance 600- and 800-MHz spectrometers equipped with cryogenically cooled probes. HSQC data set was acquired with 2048 complex points in the 1 H dimension and 160 complex points in the 15 N dimension. DHA, dissolved in the same NMR buffer, was stepwise titrated into the FATP3-TMD sample. HSQC spectra were recorded at after each DHA addition. Results The expression 、 purification and in vitro reconstitution of FATP3-TMD Figure 1a provides an overview of the workflow for the expression and purification of FATP3-TMD. Robust expression of TrpLE-FATP3-TMD fusion protein was obtained in E. coli BL21 (DE3) by induction with 0.2 mM IPTG for 16 hours at 20 ℃. The fusion was captured on Ni-NTA resin, the TrpLE tag was removed by cyanogen bromide cleavage, and the liberated peptide was polished by RP-HPLC purification (Figure 1b). SDS-PAGE (Figure 1c) and mass spectrometry confirmed the identity and purity of the product (Figure 1d, theoretical mass, 3580.59 Da; observed mass, 3580.91 Da). Following reconstitution into Fos-choline-14 micelles or DMPC/DH 6 PC bicelles, the 1 H- 15 N HSQC spectra exhibited sharp, well-dispersed cross peaks (Figures 2a and 2b), indicating that FATP3-TMD is properly folded and conformationally homogeneous, prerequisites for quantitative analyses of its interaction with DHA. Investigate the protein-ligand interaction in distinct membrane-mimetic systems HSQC spectra reveal that FATP3-TMD reconstituted in DMPC/DH 6 PC bicelles (Fig. 2b) displays more resonances with sharper, uniformly dispersed line shapes than the proten refolded in Fos-choline-14 micelles (Fig. 2a), indicating superior folding and oligomeric homogeneity in the bilayer-like bicelle matrix. To probe ligand binding, DHA was titrated into each sample at molar ratios of 0, 0.5, 1, 2, 4, and 16 relative to the protein. HSQC overlays collected after each addition (Figs. 2c, 2d) show that, in micelles, the spectra remain virtually unchanged across the entire titration series, demonstrating an absence of detectable of DHA interaction. In contrast, bicelle-reconstituted FATP3-TMD exhibits progressive, residue-specific chemical-shift perturbations as the DHA concentration increases. Twelve resonances displayed clear chemical-shift perturbations, with five showing the most pronounced changes (arrow pointed in Fig. 2d), while the reminder of the spectrum is largely conserved, confirming that the observed perturbations arise from direct DHA binding rather than nonspecific environmental effects. These results indicate that a bilayer-like bicelle environment is essential for productive recognition of DHA by FATP3-TMD, whereas detergent micelles are insufficient to support this interaction. Discussion Membrane proteins are often expressed at low levels, accumulate as aggregates or inclusion bodies, and may be cytotoxic to the host. To circumvent these challenges, we fused the FATP3 transmembrane domain to the TrpLE leader peptide, promoting high level expression in E.coli as inclusion bodies and thereby minimizing cellular toxicity. The fusion protein was captured by Ni-NTA affinity chromatography, after which the TrpLE tag was removed by cyanogen bromide (CNBr) cleavage at engineered methionine sites, a chemical strategy that obviates enzymatic digestion and eliminates labor-intensive refolding steps. This streamlined workflow yields highly pure FATP3-TMD in milligram quantities: 1 L culture in M9 minimal medium affords a uniformly 15 N labeled sample at ~0.5 mM concentration (after concentrated to 320 µl), with the entire procedure completed in only a few days. Successful in vitro studies of membrane proteins depends on reconstituting them in membrane-mimetic systems that preserve native folding and activity, yet different membrane mimetics can influence conformation and function to markedly different extents. Here, we compared the behavirour of FATP3-TMD in detergent micelles versus lipid bicelles. HSQC spectra show that bicelle-reconstituted FATP3-TMD exhibits a larger number of well-resolved, uniformly shaped resonances than the micelle sample, indicating superior structural homegeneity. Consistent with this, 1 H- 15 N NMR titrations reveal DHA-dependent chemical-shift perturbations only in the bicelle environment, demonstrating that productive ligand binding requires a bilayer-like context. Lipid molecules in bicelles provide lateral pressure, hydrophobic matching, and specific head-group contacts that stabilize the native fold and create a physiologically relevant interface for fatty acid recognition (Dai et al. 2021). By contrast, spherical detergent micelles lack a true bilayer, often forcing helices into nonnative packing and suppressing functional interactions. Bicelles have therefore become the medium of choice for structural studies of diverse membrane proteins, including G protein-coupled receptors, ion channels, and antimicrobial peptides (Corradi et al. 2019), and our results underscore their suitability for elucidating the structure-function relationships of FATP3-TMD. Future work will employ uniformly 13 C/ 15 N labelled FATP3-TMD to complete backbone resonance assignments, enabling residue-level structural mapping. Site-directed mutagenesis of candidate contact sites, followed by NMR and functional assays, will pinpoint the amino acids that mediate with DHA recognition. Together, these approaches will refine the structural model of FATP3-TMD and clarify the molecular mechanism by which FATP3 facilitates DHA transport.. In addition, we will systematically investigate how FATP3-TMD recognizes fatty acids differing in chain length and degree of unsaturation. Insights from these studies will guide the investigation of full-length FATP3, enabling more comprehensive structural and functional analyses and providing a mechanistic framework for future work on the entire FATP family. Declarations Additional Declarations: No competing interests reported. Author Contributions The manuscript was designed and written through contributions of all authors. YD, YW and WC designed the experiments. YD performed the experiments. WC supervised the entire research. All authors have given approval to the final version of the manuscript. Funding This study was funded by the National Key R&D Program of China (2022YFC2104905). Data Availability Data can be provided if there is a reasonable demand. Competing Interests The authors declare no competing interests. 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Nat Protoc 14 (8):2483-2520. http://doi.org/10.1038/s41596-019-0188-9 Gimeno RE (2007) Fatty acid transport proteins. Curr Opin Lipidol 18 (3):271-276. http://doi.org/10.1097/MOL.0b013e3281338558 Hagberg C, Mehlem A, Falkevall A, Muhl L, Eriksson U (2013) Endothelial fatty acid transport: role of vascular endothelial growth factor B. Physiology (Bethesda) 28 (2):125-134. http://doi.org/10.1152/physiol.00042.2012 Hirsch D, Stahl A, Lodish HF (1998) A family of fatty acid transporters conserved from mycobacterium to man. Proc Natl Acad Sci U S A 95 (15):8625-8629. http://doi.org/10.1073/pnas.95.15.8625 Kappen C, Kruger C, Jones S, Salbaum JM (2022) Nutrient transporter gene expression in the early conceptus-implications from two mouse models of diabetic pregnancy. Front Cell Dev Biol 10:777844. http://doi.org/10.3389/fcell.2022.777844 Koletzko B, Larqué E, Demmelmair H (2007) Placental transfer of long-chain polyunsaturated fatty acids (LC-PUFA). J Perinat Med 35 Suppl 1:S5-11. http://doi.org/10.1515/jpm.2007.030 Korbecki J, Kojder K, Jeżewski D, Simińska D, Tomasiak P, Tarnowski M, Chlubek D, Baranowska-Bosiacka I (2023) Reduced expression of very-long-chain acyl-CoA synthetases SLC27A4 and SLC27A6 in the glioblastoma tumor compared to the peritumoral area. Brain Sciences 13 (5). http://doi.org/10.3390/brainsci13050771 Maekawa M, Iwayama Y, Ohnishi T, Toyoshima M, Shimamoto C, Hisano Y, Toyota T, Balan S, Matsuzaki H, Iwata Y, Takagai S, Yamada K, Ota M, Fukuchi S, Okada Y, Akamatsu W, Tsujii M, Kojima N, Owada Y, Okano H, Mori N, Yoshikawa T (2015) Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism. Scientific Reports 5 (1):16239. http://doi.org/10.1038/srep16239 Ochiai Y, Uchida Y, Ohtsuki S, Tachikawa M, Aizawa S, Terasaki T (2017) The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport. J Neurochem 141 (3):400-412. http://doi.org/10.1111/jnc.13943 Pei Z, Fraisl P, Shi X, Gabrielson E, Forss-Petter S, Berger J, Watkins PA (2013) Very long-chain acyl-CoA synthetase 3: overexpression and growth dependence in lung cancer. PLoS One 8 (7):e69392. http://doi.org/10.1371/journal.pone.0069392 Stahl A (2004) A current review of fatty acid transport proteins (SLC27). Pflugers Arch 447 (5):722-727. http://doi.org/10.1007/s00424-003-1106-z Wood CAP, Zhang J, Aydin D, Xu Y, Andreone BJ, Langen UH, Dror RO, Gu C, Feng L (2021) Structure and mechanism of blood-brain-barrier lipid transporter MFSD2A. Nature 596 (7872):444-448. http://doi.org/10.1038/s41586-021-03782-y Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 15 Sep, 2025 Read the published version in The Journal of Membrane Biology → Version 1 posted Editorial decision: Revision requested 24 Jun, 2025 Reviews received at journal 21 Jun, 2025 Reviews received at journal 17 Jun, 2025 Reviewers agreed at journal 15 Jun, 2025 Reviewers agreed at journal 12 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers invited by journal 11 Jun, 2025 Editor assigned by journal 10 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 10 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6860198","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":470550325,"identity":"54cff9a5-3c1b-429e-9b0e-14807b096961","order_by":0,"name":"Yi Ding","email":"","orcid":"","institution":"South China University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Ding","suffix":""},{"id":470550326,"identity":"54d932bd-452e-4655-9e8f-5e810bd0e90a","order_by":1,"name":"Yonghua Wang","email":"","orcid":"","institution":"South China University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yonghua","middleName":"","lastName":"Wang","suffix":""},{"id":470550327,"identity":"d889db8e-9e87-40ce-bcc6-fd36d9fb32d4","order_by":2,"name":"Wen Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYJACZiCWQ2ITqcUYiBkbSNKS2EC0FoPjvQc/F1TcSZ/ffsb8AUOFdWID+9kD+LWcOZcsPePMs9wNZ3IMGxjOpCc28OQl4NVidiPHjJm37XDuBgkewwbGtsOJDRI8Bvi13H8D1PLvcLr8DJCWf8RoucED1NJwOIHhBkhLAxFa7M/kGEvzHDtsuOFMWuGMhGPpxm08Ofi1SLafMfzMU3NYXr798IYPH2qsZfvZz+DXggoSgJiNBPWjYBSMglEwCnAAAEyjQ+l0Po0JAAAAAElFTkSuQmCC","orcid":"","institution":"South China University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Wen","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-06-10 07:23:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6860198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6860198/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00232-025-00361-4","type":"published","date":"2025-09-15T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84574920,"identity":"a9d6932f-847d-4eed-9f8c-0199ac500fb6","added_by":"auto","created_at":"2025-06-13 16:26:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":128875,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and purification of FATP3-TMD. (a) Flowchart for the expression and purification of FATP3-TMD. (b) HPLC chromatogram of cyanogen bromide digested mix, monitored at 214 nm. (c) SDS-PAGE of FATP3-TMD, lane1: TrpLE-FATP3-TMDfusion protein captured by Ni-NTA affinity purification, lane2: Cyanogen bromide cleavage of TrpLE-FATP3-TMD fusion protein, lane3: FATP3-TMD purified by RP-HPLC. (d) Mass chromatogram of FATP3-TMD. Theoretical mass is 3580.59 Da while the measured mass is 3580.91 Da.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6860198/v1/35af818be455b5b6329f0403.png"},{"id":84574922,"identity":"64a54fcf-f0c1-422e-8276-ea860f4435e7","added_by":"auto","created_at":"2025-06-13 16:26:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140609,"visible":true,"origin":"","legend":"\u003cp\u003eReconstitution of FATP3-TMD and DHA titration. (a) \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC of FATP3-TMD reconstituted in Fos-choline-14. (b) \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC of FATP3-TMD reconstituted in DMPC/DH\u003csub\u003e6\u003c/sub\u003ePC bicelle. (c) DHA titration series for FATP3-TMD reconstituted in Fos-choline-14, overlaid HSQC spectra of sequential addition of DHA at molar ratios of 0 : 1 (red), 0.5 : 1 (blue), 1 : 1 (green), 2 : 1 (purple), 4 : 1 (pink), and 16 : 1 (gold) relative to the protein. (d) DHA titration series for FATP3-TMD reconstituted in DMPC and DH\u003csub\u003e6\u003c/sub\u003ePC, overlaid HSQC spectra of sequential addition of DHA at molar ratios of 0 : 1 (red), 0.5 : 1 (blue), 1 : 1 (green), 2 : 1 (purple), 4 : 1 (pink), and 16 : 1 (gold) relative to the protein.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6860198/v1/379e957af932c5641838262f.png"},{"id":91889880,"identity":"09014461-2105-441d-98ea-ca71aed1a4c1","added_by":"auto","created_at":"2025-09-22 16:03:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":687586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6860198/v1/60da3ea5-31be-4aec-8371-777fe3da4f56.pdf"},{"id":84574921,"identity":"ab50fa34-c1f9-4463-9847-9c4d9d4470cf","added_by":"auto","created_at":"2025-06-13 16:26:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15710,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6860198/v1/fff14dbccf8ac96b9d625234.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bilayer-Dependent Recognition of Docosahexaenoic Acid by the Transmembrane Domain of FATP3","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFatty acid transport protein 3 (FATP3), also known as very long-chain acyl-CoA synthetase 3 (ACSVL3), encoded by the gene \u003cem\u003eSLC27A3\u003c/em\u003e, is a member of the solute carrier family 27 (Hirsch et al. 1998). Sequence analysis of FATP3 reveals two conserved functional motifs: an ATP/AMP binding domain (~100 amino acids) and a FATP/VLACS-specific motif (~50 amino acids) unique to the FATP family\u0026nbsp;(DiRusso et al. 2008). FATP3 plays dual roles in lipid metabolism: (1) as an acyl-CoA synthetase, it catalyzes the activation of long-chain and very long-chain fatty acids into acyl-CoA derivatives in an ATP-dependent manner, and (2) it facilitates fatty acid update by enhancing cellular membrane permeability to fatty acids\u0026nbsp;(Pei et al. 2013). However, the extent to which FATP3 functions as a direct transporter remains a subject of ongoing investigation and debate.\u003c/p\u003e\n\u003cp\u003eFATP3 is a membrane-associated protein that contains hydrophobic transmembrane regions within its primary structure. Due to its high sequence with FATP1 (Gimeno 2007), and based on epitope tag assay of FATP1 (Stahl 2004), FATP3 is hypothesized to adopt a similar membrane topology—featuring an N-terminal transmembrane anchor and a C-terminal domain oriented toward the cytoplasm. In addition to the primary transmembrane helix, FATP3 may possess additional membrane-associating regions that facilitate its localization to the endoplasmic reticulum or other membrane compartments\u0026nbsp;(Stahl 2004). To date, no high-resolution structures of FATP3 have been reported, and its precise structural conformation remains to be experimentally determined.\u003c/p\u003e\n\u003cp\u003eHighly unsaturated ω-3 fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are essential for the development and function of neural and retinal tissues. And the mechanisms underlying their transmembrane transport have garnered significant research interest in recent years (Koletzko et al. 2007; Ochiai et al. 2017; Wood et al. 2021). FATP3 has been proposed as a potential mediator of ω-3 fatty acid transport for its expression in barrier tissues (Hagberg et al. 2013; Kappen et al. 2022). For instance, FATP3 is expressed in the placental trophoblasts and in the capillary endothelium of the blood-brain barrier, suggesting a possible role in the transfer of maternal fatty acids to the fetal brain (Maekawa et al. 2015). Although FATP3 exhibits lower expression levels in the adult brain compared to the brain-specific transporter MFSD2A—which facilitates DHA uptake in the form of lysophosphatidylcholine (Lyso-PLA)\u0026nbsp;(Korbecki et al. 2023)\u0026nbsp;—its expression is markedly higher in the embryonic mouse brain and declines after birth\u0026nbsp;(Maekawa et al. 2015). This developmental expression pattern implies that FATP3 may be involved in the provision of essential fatty acids or in membrane lipid synthesis during critical stages of neurodevelopment.\u003c/p\u003e\n\u003cp\u003eCurrently, however, direct functional evidence demonstrating that FATP3 specifically facilitates the uptake of DHA or EPA remains scarce. Indirect studies in brain microvascular endothelial cells show that, fatty acid-binding protein FABP5 binds and delivers DHA intracellularly, while both FATP3 and FATP4 are required for effective transendothelial fatty acid flux (Hagberg et al. 2013). Nontheless, there is no conclusive evidence that FATP3 mediates the transmembrane transport of ω-3 fatty acids with high specificity. Its proposed role in ω-3 fatty acid trafficking is largely inferred from tissue-specific expression patterns and indirect functional correlations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo date, FATP3 has not been examined at the molecular level. Because its extracellular domain consists of only two residues, insufficient for high-affinity binding of free fatty acids. We hypothesized that the transmembrane domain of FATP3 mediates fatty acid recognition. We therefore expressed the FATP3-TMD in \u003cem\u003eEscherichia coli\u003c/em\u003e as a TrpLE fusion, a strategy that drove high-level accumulation in inclusion bodies and enabled purification of milligram quantities of highly pure protein (Chen and Cotten 2014; Fu et al. 2019). After cyanogen-bromide cleavage and RP-HPLC purification, FATP3-TMD was reconstituted into membrane-mimetic systems, where it adopted the expected α-helical fold and retained functional integrity. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR titrations demonstrated specific binding of ω-3 polyunsaturated fatty acids such as DHA to FATP3-TMD, providing a molecular foundation for elucidating how FATP3 facilitates DHA transport across biological membranes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eDetailed materials and methods can be found in the supplemantary documents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of recombinant plasmid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequence encoding residues 1-32 of human FATP3 (UniProt Q8N163: MAALLLLPLLLLLPLLLLKLHLWPQLRWLPAD) was codon-optimized for \u003cem\u003eE. coli\u003c/em\u003e expression, Synthesized de novo, and subcloned into the pMM-LR6 vector to generate the recombinant expression plasmid pMM-LR6-FATP3-TMD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and isotopic labeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eBL21 (DE3) cells harbouring pMM-LR6-FATP3-TMD were grown in LB medium (50 \u0026micro;g/mL kanamycin) until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6-0.7. Expression was induced with 0.2 mM IPTG, and the culture was shifted to 20℃ for 16 hours. Cells were collected by centrifugation and stored at -80℃. Uniformly \u003csup\u003e15\u003c/sup\u003eN labeled FATP3-TMD was produced under identical conditions in M9 minimal medium supplemented with \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (1 g/L) as the sole nitrogen source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial pellets were\u0026nbsp;disrupted by sonication.\u0026nbsp;Soluble fractions were removed by centrifugation, and the inclusion-body pellet was retained and solubilized in the guanidine buffer. Insoluble material was removed by a second centrifugation step.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe supernatant was initially purified by Ni-NTA resin. Cyanogen bromide (CNBr) was added to the eluate and cleavage was carried out. The reaction mixture was transferred to 2 kDa MWCO dialysis tubing and dialyzed twice against ultrapure water, then lyophilized.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dried material was dissolved in 50 % (v/v) formic acid, then purified by RP-HPLC on a C3 column (Agilent, ZORBAX) using an isopropanol/acetonitrile/water gradient containing 0.1 % trifluoroacetic acid. Fractions containing the target FATP3-TMD were pooled, lyophilised, and stored at -20 ℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular weight of the purified FATP3-TMD was confirmed by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReconstitution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicelles: Fos-Choline-14 and Lyophilized FATP3-TMD was dissolved and gently mixed in 6 M guanidine in NMR buffer (25 mM MES, pH 6.7). The solution was transferred to a 2 KDa MWCO dialysis cassette (Thermo Scientific) and dialyzed against detergent-free NMR buffer to allow micelle formation and refolding. The dialysate was concentrated to 300 \u0026mu;l by centrifugation, and supplemented with 20 \u0026mu;l of D\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\n\u003cp\u003eBicelles (\u003cem\u003eq\u0026nbsp;\u003c/em\u003e~0.4): DMPC, DH\u003csub\u003e6\u003c/sub\u003ePC and protein sample were co-dissolved in hexafluoroisopropanol. The solvent was evaporated under gentle nitrogen stream, and the mixture was further dried under vacuum. The residue was rehydrated in 6 M guanidine in NMR buffer, and was dialyzed as above to remove denaturant and allow bicelle assembly. After dialysis, the sample was clarified, concentrated to 300 \u0026micro;l, supplemented with 20 \u0026micro;l of D\u003csub\u003e2\u003c/sub\u003eO, and transferred to a Shigemi tube for NMR measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMR titration experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e1\u003c/sup\u003eH heteronuclear single-quantum coherence (HSQC) spectra were recorded at 35 ℃ on Bruker Avance 600- and 800-MHz spectrometers equipped with cryogenically cooled probes. HSQC data set was acquired with 2048 complex points in the \u003csup\u003e1\u003c/sup\u003eH dimension and 160 complex points in the \u003csup\u003e15\u003c/sup\u003eN dimension. DHA, dissolved in the same NMR buffer, was stepwise titrated into the FATP3-TMD sample. HSQC spectra were recorded at after each DHA addition.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e \u003cstrong\u003eexpression\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003epurification and in vitro reconstitution of FATP3-TMD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 1a provides an overview of the workflow for the expression and purification of FATP3-TMD. Robust expression of TrpLE-FATP3-TMD fusion protein was obtained in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) by induction with 0.2 mM IPTG for 16 hours at 20\u0026nbsp;℃. The fusion was captured on Ni-NTA resin, the TrpLE tag was removed by cyanogen bromide cleavage, and the liberated peptide was polished by RP-HPLC purification (Figure 1b). SDS-PAGE (Figure 1c) and mass spectrometry confirmed the identity and purity of the product (Figure 1d, theoretical mass, 3580.59 Da; observed mass, 3580.91 Da).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing reconstitution into Fos-choline-14 micelles or DMPC/DH\u003csub\u003e6\u003c/sub\u003ePC bicelles, the \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra exhibited sharp, well-dispersed cross peaks (Figures 2a and 2b), indicating that FATP3-TMD is properly folded and conformationally homogeneous, prerequisites for quantitative analyses of its interaction with DHA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigate the protein-ligand interaction in distinct membrane-mimetic systems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHSQC spectra reveal that FATP3-TMD reconstituted in DMPC/DH\u003csub\u003e6\u003c/sub\u003ePC bicelles (Fig. 2b) displays more resonances with sharper, uniformly dispersed line shapes than the proten refolded in Fos-choline-14 micelles (Fig. 2a), indicating superior folding and oligomeric homogeneity in the bilayer-like bicelle matrix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo probe ligand binding, DHA was titrated into each sample at molar ratios of 0, 0.5, 1, 2, 4, and 16 relative to the protein. HSQC overlays collected after each addition (Figs. 2c, 2d) show that, in micelles, the spectra remain virtually unchanged across the entire titration series, demonstrating an absence of detectable of DHA interaction. In contrast, bicelle-reconstituted FATP3-TMD exhibits progressive, residue-specific chemical-shift perturbations as the DHA concentration increases. Twelve resonances displayed clear chemical-shift perturbations, with five showing the most pronounced changes (arrow pointed in Fig. 2d), while the reminder of the spectrum is largely conserved, confirming that the observed perturbations arise from direct DHA binding rather than nonspecific environmental effects. These results indicate that a bilayer-like bicelle environment is essential for productive recognition of DHA by FATP3-TMD, whereas detergent micelles are insufficient to support this interaction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMembrane proteins are often expressed at low levels, accumulate as aggregates or inclusion bodies, and may be cytotoxic to the host. To circumvent these challenges, we fused the FATP3 transmembrane domain to the TrpLE leader peptide, promoting high level expression in\u003cem\u003e\u0026nbsp;E.coli\u003c/em\u003e as inclusion bodies and thereby minimizing cellular toxicity. The fusion protein was captured by Ni-NTA affinity chromatography, after which the TrpLE tag was removed by cyanogen bromide (CNBr) cleavage at engineered methionine sites, a chemical strategy that obviates enzymatic digestion and eliminates labor-intensive refolding steps. This streamlined workflow yields highly pure FATP3-TMD in milligram quantities: 1 L culture in M9 minimal medium affords a uniformly \u003csup\u003e15\u003c/sup\u003eN labeled sample at ~0.5 mM concentration (after concentrated to 320 µl), with the entire procedure completed in only a few days.\u003c/p\u003e\n\u003cp\u003eSuccessful in vitro studies of membrane proteins depends on reconstituting them in membrane-mimetic systems that preserve native folding and activity, yet different membrane mimetics can influence conformation and function to markedly different extents. Here, we compared the behavirour of FATP3-TMD in detergent micelles versus lipid bicelles. HSQC spectra show that bicelle-reconstituted FATP3-TMD exhibits a larger number of well-resolved, uniformly shaped resonances than the micelle sample, indicating superior structural homegeneity. Consistent with this, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR titrations reveal DHA-dependent chemical-shift perturbations only in the bicelle environment, demonstrating that productive ligand binding requires a bilayer-like context. Lipid molecules in bicelles provide lateral pressure, hydrophobic matching, and specific head-group contacts that stabilize the native fold and create a physiologically relevant interface for fatty acid recognition (Dai et al. 2021). By contrast, spherical detergent micelles lack a true bilayer, often forcing helices into nonnative packing and suppressing functional interactions. Bicelles have therefore become the medium of choice for structural studies of diverse membrane proteins, including G protein-coupled receptors, ion channels, and antimicrobial peptides (Corradi et al. 2019), and our results underscore their suitability for elucidating the structure-function relationships of FATP3-TMD.\u003c/p\u003e\n\u003cp\u003eFuture work will employ uniformly \u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e15\u003c/sup\u003eN labelled FATP3-TMD to complete backbone resonance assignments, enabling residue-level structural mapping. Site-directed mutagenesis of candidate contact sites, followed by NMR and functional assays, will pinpoint the amino acids that mediate with DHA recognition. Together, these approaches will refine the structural model of FATP3-TMD and clarify the molecular mechanism by which FATP3 facilitates DHA transport..\u003c/p\u003e\n\u003cp\u003eIn addition, we will systematically investigate how FATP3-TMD recognizes fatty acids differing in chain length and degree of unsaturation. Insights from these studies will guide the investigation of full-length FATP3, enabling more comprehensive structural and functional analyses and providing a mechanistic framework for future work on the entire FATP family.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAdditional Declarations:\u003c/strong\u003e No competing interests reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eThe manuscript was designed and written through contributions of all authors. YD, YW and WC designed the experiments. YD performed the experiments. WC supervised the entire research. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was funded by the National Key R\u0026amp;D Program of China (2022YFC2104905).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e Data can be provided if there is a reasonable demand.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen W, Cotten ML (2014) Expression, purification, and micelle reconstitution of antimicrobial piscidin 1 and piscidin 3 for NMR studies. Protein Expr Purif 102:63-68. http://doi.org/10.1016/j.pep.2014.08.001\u003c/li\u003e\n\u003cli\u003eCorradi V, Sejdiu BI, Mesa-Galloso H, Abdizadeh H, Noskov SY, Marrink SJ, Tieleman DP (2019) Emerging Diversity in Lipid\u0026ndash;Protein Interactions. Chemical Reviews 119 (9):5775-5848. http://doi.org/10.1021/acs.chemrev.8b00451\u003c/li\u003e\n\u003cli\u003eDai Y, Tang H, Pang S (2021) The Crucial Roles of Phospholipids in Aging and Lifespan Regulation. Front Physiol 12:775648. http://doi.org/10.3389/fphys.2021.775648\u003c/li\u003e\n\u003cli\u003eDiRusso CC, Darwis D, Obermeyer T, Black PN (2008) Functional domains of the fatty acid transport proteins: studies using protein chimeras. Biochim Biophys Acta 1781 (3):135-143. http://doi.org/10.1016/j.bbalip.2008.01.002\u003c/li\u003e\n\u003cli\u003eFu Q, Piai A, Chen W, Xia K, Chou JJ (2019) Structure determination protocol for transmembrane domain oligomers. Nat Protoc 14 (8):2483-2520. http://doi.org/10.1038/s41596-019-0188-9\u003c/li\u003e\n\u003cli\u003eGimeno RE (2007) Fatty acid transport proteins. Curr Opin Lipidol 18 (3):271-276. http://doi.org/10.1097/MOL.0b013e3281338558\u003c/li\u003e\n\u003cli\u003eHagberg C, Mehlem A, Falkevall A, Muhl L, Eriksson U (2013) Endothelial fatty acid transport: role of vascular endothelial growth factor B. Physiology (Bethesda) 28 (2):125-134. http://doi.org/10.1152/physiol.00042.2012\u003c/li\u003e\n\u003cli\u003eHirsch D, Stahl A, Lodish HF (1998) A family of fatty acid transporters conserved from mycobacterium to man. Proc Natl Acad Sci U S A 95 (15):8625-8629. http://doi.org/10.1073/pnas.95.15.8625\u003c/li\u003e\n\u003cli\u003eKappen C, Kruger C, Jones S, Salbaum JM (2022) Nutrient transporter gene expression in the early conceptus-implications from two mouse models of diabetic pregnancy. Front Cell Dev Biol 10:777844. http://doi.org/10.3389/fcell.2022.777844\u003c/li\u003e\n\u003cli\u003eKoletzko B, Larqu\u0026eacute; E, Demmelmair H (2007) Placental transfer of long-chain polyunsaturated fatty acids (LC-PUFA). J Perinat Med 35 Suppl 1:S5-11. http://doi.org/10.1515/jpm.2007.030\u003c/li\u003e\n\u003cli\u003eKorbecki J, Kojder K, Jeżewski D, Simińska D, Tomasiak P, Tarnowski M, Chlubek D, Baranowska-Bosiacka I (2023) Reduced expression of very-long-chain acyl-CoA synthetases SLC27A4 and SLC27A6 in the glioblastoma tumor compared to the peritumoral area. Brain Sciences 13 (5). http://doi.org/10.3390/brainsci13050771\u003c/li\u003e\n\u003cli\u003eMaekawa M, Iwayama Y, Ohnishi T, Toyoshima M, Shimamoto C, Hisano Y, Toyota T, Balan S, Matsuzaki H, Iwata Y, Takagai S, Yamada K, Ota M, Fukuchi S, Okada Y, Akamatsu W, Tsujii M, Kojima N, Owada Y, Okano H, Mori N, Yoshikawa T (2015) Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism. Scientific Reports 5 (1):16239. http://doi.org/10.1038/srep16239\u003c/li\u003e\n\u003cli\u003eOchiai Y, Uchida Y, Ohtsuki S, Tachikawa M, Aizawa S, Terasaki T (2017) The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport. J Neurochem 141 (3):400-412. http://doi.org/10.1111/jnc.13943\u003c/li\u003e\n\u003cli\u003ePei Z, Fraisl P, Shi X, Gabrielson E, Forss-Petter S, Berger J, Watkins PA (2013) Very long-chain acyl-CoA synthetase 3: overexpression and growth dependence in lung cancer. PLoS One 8 (7):e69392. http://doi.org/10.1371/journal.pone.0069392\u003c/li\u003e\n\u003cli\u003eStahl A (2004) A current review of fatty acid transport proteins (SLC27). Pflugers Arch 447 (5):722-727. http://doi.org/10.1007/s00424-003-1106-z\u003c/li\u003e\n\u003cli\u003eWood CAP, Zhang J, Aydin D, Xu Y, Andreone BJ, Langen UH, Dror RO, Gu C, Feng L (2021) Structure and mechanism of blood-brain-barrier lipid transporter MFSD2A. Nature 596 (7872):444-448. http://doi.org/10.1038/s41586-021-03782-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FATP3-TMD, DHA, Micelles, Bicelles, NMR","lastPublishedDoi":"10.21203/rs.3.rs-6860198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6860198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFatty 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 \u003cem\u003eEscherichia coli\u003c/em\u003e as a TrpLE fusion, liberated by cyanogen-bromide cleavage and polished by one-step reverse-phase HPLC, yielding milligram quantities of highly pure peptide. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN 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.\u003c/p\u003e","manuscriptTitle":"Bilayer-Dependent Recognition of Docosahexaenoic Acid by the Transmembrane Domain of FATP3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 16:26:09","doi":"10.21203/rs.3.rs-6860198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-24T15:46:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-21T21:27:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T16:49:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274527608365592457141822003874553824354","date":"2025-06-15T10:44:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217021863242969876965779748656793038472","date":"2025-06-12T19:19:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223649793456618411592369232260422130939","date":"2025-06-11T17:23:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-11T16:44:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-10T14:03:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T14:02:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Membrane Biology","date":"2025-06-10T07:21:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5dffd378-134b-4cb7-a623-5e1980603942","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T16:01:14+00:00","versionOfRecord":{"articleIdentity":"rs-6860198","link":"https://doi.org/10.1007/s00232-025-00361-4","journal":{"identity":"the-journal-of-membrane-biology","isVorOnly":false,"title":"The Journal of Membrane Biology"},"publishedOn":"2025-09-15 15:57:33","publishedOnDateReadable":"September 15th, 2025"},"versionCreatedAt":"2025-06-13 16:26:09","video":"","vorDoi":"10.1007/s00232-025-00361-4","vorDoiUrl":"https://doi.org/10.1007/s00232-025-00361-4","workflowStages":[]},"version":"v1","identity":"rs-6860198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6860198","identity":"rs-6860198","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}