Transfer of stabilising mutations between different secondary active transporter families.
Transfer of stabilising mutations between different secondary active transporter families.
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DOI：
10.1002/2211-5463.13168
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发表时间：
2021-06
期刊：
影响因子：
2.6
通讯作者：
Byrne B
中科院分区：
文献类型：
--
作者：
Cecchetti C;Scull NJ;Mohan TC;Alguel Y;Jones AMC;Cameron AD;Byrne B
膜整合转运蛋白在底物跨膜转运中起着重要的作用。产生适合于结构研究的转运蛋白的一种方法是引入降低构象灵活性并增加稳定性的突变。然而，很难预测哪些突变会导致更稳定的蛋白质。之前，我们通过引入单点突变G411 V，使蛋白质陷入内向构象，稳定了SLC 23家族成员尿酸-黄嘌呤转运蛋白UapA。在这里，我们试图稳定的结构相关的BOR 1转运蛋白拟南芥，SLC 4家族的成员，通过引入等效的取代。我们鉴定了AtBOR 1中可能的残基P362和M363，它们可能与UapA的G411相当，并产生了四个突变体P362 V或L和M363 F或Y。使用加热荧光尺寸排阻色谱法的稳定性分析表明，M363 F/Y突变体比WT AtBOR 1和P362 V/L突变体更稳定。此外，功能互补分析显示，与P362 V/L和WT蛋白相比，M363 F/Y突变体的转运活性降低。M363 F/Y蛋白的纯化和结晶产生了比WT衍射更好的晶体（5.5 μ g对7 μ g）。我们假设，大量增加的F和Y取代限制了蛋白质进行与运输相关的构象重排的能力。这些蛋白质代表了AtBOR 1未来研究的基础。我们将构巢曲霉的膜转运蛋白UapA中的稳定取代（G411 V）转移到模式植物拟南芥的结构相关的BOR 1转运蛋白中。氨基酸取代M363 F和M363 Y导致AtBOR 1的稳定性增加，与降低的转运活性相关，从而降低构象灵活性。AtBOR 1 M363可能与UapA G411相当。
Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid‐xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single‐point mutation, G411V, trapping the protein in the inward‐facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1. We transferred a stabilising substitution (G411V) in the membrane transporter UapA from Aspergillus nidulans to the structurally related BOR1 transporter from the model plant, Arabidopsis thaliana. Amino acid substitutions M363F and M363Y result in increased stability of AtBOR1 associated with reduced transport activity and thus reduced conformational flexibility. AtBOR1 M363 is likely equivalent to UapA G411.
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影响因子：
16.6
作者：
Huynh KW;Jiang J;Abuladze N;Tsirulnikov K;Kao L;Shao X;Newman D;Azimov R;Pushkin A;Zhou ZH;Kurtz I
通讯作者：
Kurtz I
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作者：
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作者：
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通讯作者：
Palacin, Manuel
影响因子：
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作者：
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通讯作者：
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