FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
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Author:
Date:
2021-04-05
[Abstract] Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then ...
[摘要] [摘要]次级活性转运蛋白驻留在细胞膜中,利用离子的电化学梯度作为能量源,可针对陡峭的浓度梯度转运极性氨基酸(如氨基酸)。通常,基于集合的放射性标记底物摄取或转运电流的测量可确定转运蛋白的动力学参数。在这里,我们描述了一种基于荧光的谷氨酸和天冬氨酸转运蛋白功能测定方法,该方法使用古细菌升降剂型钠和天冬氨酸共转运蛋白Glt Ph作为模型系统,提供了单转运蛋白,单转运周期的分辨率。我们准备包含重组的纯化的Glt Ph转运蛋白和封装的周质谷氨酸/天冬氨酸结合蛋白,PEB1a,用供体和受体荧光团标记的蛋白脂质体。然后,我们将蛋白脂质体表面固定化,并使用单分子全内反射荧光(TIRF)显微镜测量随时间变化的运输依赖性荧光共振能量转移(FRET)效率变化。与放射性配体摄取测定法相比,该测定法在时间分辨率上提高了10-100倍。它还可以对不同转运周期步骤进行动力学表征,并识别转运蛋白种群内的动力学异质性。
[背景]膜驻留的二级主动转运蛋白或溶质载体(SLC)介导氨基酸,激素,神经递质,维生素和药物等溶质的细胞摄取。他们将集中的底物摄取与主要通过Na + / K + ATPases的作用维持的离子电化学梯度的能量上有利的耗散结合在一起(Lingrel and Kuntzweiler ...
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Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example
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Author:
Date:
2017-09-05
[Abstract] The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating ...
[摘要] 与共激活因子p300的转录因子缺氧诱导因子(HIF)复合物,激活缺氧反应途径并允许肿瘤生长。每个相应蛋白质的CH1和CAD结构域形成p300和HIF之间的界面。正在开发靶向和抑制HIF / p300复合物形成的小分子化合物,目的是减少肿瘤生长。研究配体与p300-CH1相互作用的高分辨NMR光谱是必要的,为了进行结构和生物物理学研究,需要净化大量正确折叠的p300-CH1。 p300-CH1具有3个锌指和9个半胱氨酸残基,构成与试剂相容性和蛋白氧化相关的挑战。已经开发了一种通过在表达过程中并入锌并简化纯化时间来克服这些问题的方案,导致适合于结构NMR研究的最佳折叠蛋白质(120mg / 4L表达介质)的高产率。已证实最终重组p300-CH1的结构完整性是使用一维1 H NMR光谱和圆二色性最优的。该方案适用于纯化其他含锌指蛋白质。 【背景】由于不适当的血管灌注,实体瘤的发展与缺氧区的发展有关。对于缺氧微环境,肿瘤细胞过表达低氧诱导因子(HIF),一种异二聚体转录因子家族(Semenza,2002; Brat和Van Meir,2004; Kaur等,2005)。 HIFs结合p300(一种转录共激活因子),形成诱导HIF靶基因的复合物,从而激活缺氧反应途径并促进肿瘤生长(Kasper and Brindle,2006; Liu,2008)。涉及HIF / p300蛋白 ...
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