美国科学家开发出一种利用微小荧光分子快速发现和识别活细胞中蛋白质相互作用的新技术。该技术避免了旧方法中可能产生生物破坏的缺陷,相关内容发表在新出版的《自然:化学生物》杂志上。
通常,人们利用不同的绿荧光蛋白质(GFP)来为其它蛋白质做标识。但是绿荧光蛋白质不仅大,而且对许多活细胞具有毒性,因此难以用于研究活细胞。此外,绿荧光蛋白质还往往会自动聚集,让研究人员不容易利用和观察它们。
美国耶鲁大学化学教授阿兰娜.谢葩紫领导的研究小组利用名为“profluorescent”的荧光小分子而不是荧光蛋白质,开发出了新的标识技术。荧光小分子能十分容易地进入活细胞,并在蛋白质内与氨基酸标识序列“捆绑”后发出荧光。新技术让研究人员能够更准确地了解单个活细胞中独立蛋白质交迭区复杂的接触以及各蛋白质之间的关系。
每个蛋白质由于其直线氨基酸链发出交迭而呈现三维结构。通常每种蛋白质只有一种具有工作能力的形状,蛋白质这种特殊形状的产生取决于其氨基酸和细胞中的其它过程。研究人员表示,经过对所研究的蛋白质和其氨基酸链经过处理,当蛋白质交迭正确并出现特定的氨基酸标识序列后,它对荧光小分子具有强烈的亲合力,能够吸引并“捆绑”上较多的荧光小分子,发出明亮的荧光(见左图上半部分);如果蛋白质交迭错误,那么其吸引和“捆绑”染色小分子的能力较差,所发荧光十分暗淡(见左图下半部分)。
虽然这些能够“捆绑”单个蛋白质的荧光小分子化合物已被使用了10年,但是这是研究人员首次将其用于识别蛋白质间的相互作用。谢葩紫表示,新的标识方法为了解细胞中蛋白质如何选择其伙伴提供了重要的观察手段,这与人们在试管中所观察到的也许具有相当大的差别。
谢葩紫同时认为,从理论上讲,新标识技术有望作为疗法有选择性地阻止细胞中某些特殊蛋白质的活动,或者作为诊断方法,为人们提供细胞内蛋白质的高清晰可视结构图。她预计,新技术可能应用于识别神经退化疾病(如帕金森病)患者细胞中蛋白质发生的交迭错误。(科技日报)
原始出处:
Nature Chemical Biology
Published online: 4 November 2007 | doi:10.1038/nchembio.2007.49
Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH
Nathan W Luedtke1,3, Rachel J Dexter1, Daniel B Fried1 & Alanna Schepartz1,2
Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide–biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.
Schematic of fluorescent detector: When a target protein is folded correctly, "tags" come together so that the dye binds with high affinity and fluoresces brightly; misfolded proteins have low affinity for the dye. (Credit: Schepartz/Nature Chemical Biology)
Department of Chemistry, Yale University, 225 Prospect Street, New Haven, Connecticut 06520-8107, USA.
Department of Chemistry and Molecular, Cellular, and Developmental Biology, Yale University, 219 Prospect Street, New Haven, Connecticut 06520-8107, USA.
Present address: Universität Zürich, Organisch-chemisches Institut, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Correspondence to: Alanna Schepartz1,2 Email: alanna.schepartz@yale.edu