Comparative stability of Major Facilitator Superfamily transport proteins
Comparative stability of Major Facilitator Superfamily transport proteins
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DOI：
10.1007/s00249-017-1197-7
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发表时间：
2017-10-01
影响因子：
2
通讯作者：
Booth, Paula J.
中科院分区：
文献类型：
--
作者：
Harris, Nicola J.;Findlay, Heather E.;Booth, Paula J.
膜转运蛋白是一类重要的蛋白质，其可用的结构和热力学信息很少。主要促进子超家族 (MFS) 是一大类转运蛋白，负责转运真核生物和原核生物中的多种底物。我们使用远紫外圆二色性（CD）来评估这个超家族的转运蛋白是否具有相同的化学和热稳定性。我们比较了五种不同的 MFS 转运蛋白的稳定性；来自希瓦氏菌的 PepT(So) 和来自大肠杆菌的 LacY、GalP、GlpT 和 XylE，以及已知的 LacY 稳定突变体 LacY-C154G。 CD 稳定性测量表明这些转运蛋白分为两大类。 “尿素敏感”类别包括 LacY-WT、GalP 和 GlpT，它们在 8 M 尿素中分别丢失了约三分之一的二级结构，在更严格的变性剂盐酸胍 (GuHCl) 中丢失了三分之二。 “耐尿素”类别包括 LacY-C154G、XylE 和 PepT(So)。这些抗性转运蛋白在 8 M 尿素中损失很少的二级结构，并且 LacY-C154G 和 PepT(So) 能够抵抗浓度高达 4 M 的 GuHCl 变性。LacY、GlpT、XylE 和 PepT(So) 的稳定性与其晶体结构构象相关，这意味着在体外采用相似的构象。 “尿素敏感”转运蛋白 LacY 和 GlpT 结晶为向内开放状态，而 XylE 和 PepT(So) 结晶为封闭状态。这项研究强调了研究各种相似蛋白质的重要性，因为相似的二级结构和整体功能不一定在体外具有相同的稳定性。
Membrane transporters are a vital class of proteins for which there is little available structural and thermodynamic information. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates in eukaryotes and prokaryotes. We have used far-UV circular dichroism (CD) to assess whether transporters from this superfamily have the same chemical and thermal stability. We have compared the stability of five different MFS transporters; PepT(So) from Shewanella oneidensis and LacY, GalP, GlpT and XylE from Escherichia coli, as well as a known stable mutant of LacY, LacY-C154G. CD stability measurements revealed that these transporters fall into two broad categories. The 'urea-sensitive' category includes LacY-WT, GalP and GlpT, which each lose around a third of their secondary structure in 8 M urea and two-thirds in the harsher denaturant guanidine hydrochloride (GuHCl). The 'urea-resistant' category includes LacY-C154G, XylE and PepT(So). These resistant transporters lose very little secondary structure in 8 M urea, and LacY-C154G and PepT(So) resist denaturation by GuHCl up to a concentration of 4 M. The stabilities of LacY, GlpT, XylE and PepT(So) correlated with their crystal structure conformations, implying that a similar conformation is adopted in vitro. The 'urea-sensitive' transporters LacY and GlpT were crystallised inward-open states, while XylE and PepT(So) were crystallised in occluded states. This study highlights the importance of studying a wide range of similar proteins, as a similar secondary structure and overall function does not necessarily confer the same stability in vitro.