Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
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DOI：
10.1371/journal.pone.0078301
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Jansen M
中科院分区：
文献类型：
--
作者：
Duddempudi PK;Goyal R;Date SS;Jansen M
质子偶联叶酸转运蛋白（PCFT）最近被确定为人类膳食叶酸的主要摄取途径。PCFT的三维结构及其与功能的详细相互作用仍有待确定。我们使用取代的半胱氨酸可及性方法筛选了HsPCFT的水可及的细胞外表面，以研究水可及的表面和不可及的埋藏蛋白片段之间的边界。在功能性无半胱氨酸HsPCFT背景构建体中的40个位置处单独工程化的单半胱氨酸，用双层不渗透性伯胺反应性生物素化剂（磺基琥珀酰亚胺基6-（生物素酰胺基）己酸酯）探测爪蟾卵母细胞中的质膜表达，并且另外用巯基选择性生物素化剂2-（（生物素酰基）氨基）乙基甲硫代磺酸酯探测相应工程化半胱氨酸的水可及性。Cys选择性标记与胺选择性标记之间的比率进一步用于评估通过同源/穿线建模产生的HsPCFT的三维模型。具有已知实验确定的三维结构的HsPCFT的最接近的同源物都是最大的膜蛋白超家族之一的成员，主要易化剂超家族（MFS）。HsPCFT和这些模板之间的低序列同一性- 14%或更低-需要基于实验的评估和同源性/穿线模型的模型改进。随着目前的单半胱氨酸辅助组，基于GlpT和PepTSt的模型是最有前途的进一步完善。
The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity - 14% or less – between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology / threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.
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影响因子：
4.8
作者：
Britto, PJ;Knipling, L;Wolff, J
通讯作者：
Wolff, J
影响因子：
11.4
作者：
Mirza, O;Guan, L;Kaback, HR
通讯作者：
Kaback, HR
影响因子：
3.8
作者：
Meyer, Esther;Kurian, Manju A.;Maher, Eamonn R.
通讯作者：
Maher, Eamonn R.
DOI：
10.1073/pnas.94.7.3290
发表时间：
1997-04-01
影响因子：
11.1
作者：
Blount, BC;Mack, MM;Ames, BN
通讯作者：
Ames, BN
影响因子：
6.7
作者：
Duthie, SJ
通讯作者：
Duthie, SJ