美國科學(xué)家開發(fā)出一種利用微小熒光分子快速發(fā)現(xiàn)和識別活細(xì)胞中蛋白質(zhì)相互作用的新技術(shù)。該技術(shù)避免了舊方法中可能產(chǎn)生生物破壞的缺陷,，相關(guān)內(nèi)容發(fā)表在新出版的《自然：化學(xué)生物》雜志上,。
通常，人們利用不同的綠熒光蛋白質(zhì)（GFP）來為其它蛋白質(zhì)做標(biāo)識,。但是綠熒光蛋白質(zhì)不僅大,，而且對許多活細(xì)胞具有毒性,，因此難以用于研究活細(xì)胞,。此外,，綠熒光蛋白質(zhì)還往往會自動聚集，讓研究人員不容易利用和觀察它們,。
美國耶魯大學(xué)化學(xué)教授阿蘭娜.謝葩紫領(lǐng)導(dǎo)的研究小組利用名為“profluorescent”的熒光小分子而不是熒光蛋白質(zhì),，開發(fā)出了新的標(biāo)識技術(shù)。熒光小分子能十分容易地進(jìn)入活細(xì)胞,，并在蛋白質(zhì)內(nèi)與氨基酸標(biāo)識序列“捆綁”后發(fā)出熒光,。新技術(shù)讓研究人員能夠更準(zhǔn)確地了解單個活細(xì)胞中獨(dú)立蛋白質(zhì)交迭區(qū)復(fù)雜的接觸以及各蛋白質(zhì)之間的關(guān)系。
每個蛋白質(zhì)由于其直線氨基酸鏈發(fā)出交迭而呈現(xiàn)三維結(jié)構(gòu),。通常每種蛋白質(zhì)只有一種具有工作能力的形狀,，蛋白質(zhì)這種特殊形狀的產(chǎn)生取決于其氨基酸和細(xì)胞中的其它過程。研究人員表示,，經(jīng)過對所研究的蛋白質(zhì)和其氨基酸鏈經(jīng)過處理,，當(dāng)?shù)鞍踪|(zhì)交迭正確并出現(xiàn)特定的氨基酸標(biāo)識序列后，它對熒光小分子具有強(qiáng)烈的親合力,，能夠吸引并“捆綁”上較多的熒光小分子,，發(fā)出明亮的熒光（見左圖上半部分）；如果蛋白質(zhì)交迭錯誤,，那么其吸引和“捆綁”染色小分子的能力較差,，所發(fā)熒光十分暗淡（見左圖下半部分）。
雖然這些能夠“捆綁”單個蛋白質(zhì)的熒光小分子化合物已被使用了10年,，但是這是研究人員首次將其用于識別蛋白質(zhì)間的相互作用,。謝葩紫表示，新的標(biāo)識方法為了解細(xì)胞中蛋白質(zhì)如何選擇其伙伴提供了重要的觀察手段,，這與人們在試管中所觀察到的也許具有相當(dāng)大的差別,。
謝葩紫同時認(rèn)為，從理論上講,，新標(biāo)識技術(shù)有望作為療法有選擇性地阻止細(xì)胞中某些特殊蛋白質(zhì)的活動,，或者作為診斷方法，為人們提供細(xì)胞內(nèi)蛋白質(zhì)的高清晰可視結(jié)構(gòu)圖,。她預(yù)計,，新技術(shù)可能應(yīng)用于識別神經(jīng)退化疾病（如帕金森?。┗颊呒?xì)胞中蛋白質(zhì)發(fā)生的交迭錯誤,。（科技日報）
原始出處:
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: [email protected]
