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      您的位置: 首頁>BLOGS>研究進展>GFP亮不亮,納米抗體說了算

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      GFP亮不亮,納米抗體說了算

      在細胞生物學研究中,熒光蛋白幾乎無處不在,因此科學家對它的改造也在層層升級。eGFP應該是大家最熟悉的一種增強型變異體,與野生型的GFP相比,亮度更強,光穩定性更好。然而,科學的探索是永無止境的,研究人員還在尋覓更好的熒光蛋白。這不,德國慕尼黑大學的Ulrich Rothbauer和他的同事就另辟蹊徑,采用一種獨特的方法來改變GFP的特性。文章發表在近期的《Nature Structural & Molecular Biology》上。


      他們的秘密武器是納米抗體(nanobody)。這些來源于駱駝科動物的單結構域肽段與抗體相似,都有著與抗原結合的性質,不過它們的穩定性更好,體積更小。根據這一性質,研究人員嘗試篩選出能調節GFP構象及光譜性質的納米抗體。利用噬菌體展示篩選,他們鑒定出7種能與GFP結合的不同納米抗體。




      Figure 1 Identification of nanobodies modulating the fluorescence of GFP. (a) Fluorescence in vitro binding assay. Titration of seven unique GFP binding proteins (GBP1–7) from 0–50 nM on 50 nM purified wtGFP. The fluorescence signal intensity of wtGFP was quantified using a laser scanner. (b) Minimizer can be displaced by Enhancer but not vice versa. Upper row, GFP was either mock incubated or incubated with equimolar amounts of Enhancer, or Enhancer was added followed immediately (5–15 s) by equimolar amounts of Minimizer. Lower row, same experimental setup as above but with Minimizer being added first. GFP emission was detected as described for a. (c) Quantification of GFP fluorescence as shown in b. The order of addition of Enhancer or Minimizer is indicated by numbers 1 and 2. Means and s.d. (error bars) of three independent experiments are shown.


      其中一個納米抗體與GFP結合后,熒光比eGFP本身增強了4倍,后來他們將之命名為Enhancer。結構分析表明,在GFP-Enhancer的復合體中,發光基團帶有負電荷。而Enhancer與eGFP的結合只讓熒光增強了1.5倍,于是作者們推測Enhancer的結合行使著與eGFP的突變類似的功能,從而提高了熒光強度。實際上,Enhancer-GFP的吸收光譜與eGFP相似。

      同時,研究人員還發現了另一個納米抗體,在結合后能將GFP的熒光強度降低5倍,他們稱其為Minimizer。



      Structures of the GFP–nanobody complexes. (ad) Enhancer (a; light blue ribbon model) and Minimizer (c) (orange ribbon model) recognize two different nonlinear epitopes on the surface of the GFP a-can (green ribbon model). The insets in and show details of the binding sites with selected residues and the GFP chromophore (Cro66GFP) highlighted as sticks. The chromophore environments for the GFP–Enhancer (b) and GFP–Minimizer complexes (d), respectively, are superimposed with 2Fo ? Fc density maps (contoured at 1.0ó). Two alternative conformations of R168GFP are marked with * and **. Nanobody residues numbered as previously described19; in Minimizer, the 15 residues corresponding to position 100 are labeled a-o.


      Enhancer和Minimizer的調節不僅發生在體外,在活細胞內也能重演。作者們將Enhancer用于一系列體內應用中,包括帶GFP標簽的雌激素受體在Hela細胞中的運輸。在激素誘導下,重組子轉位到細胞核,在那里它與核定位的Enhancer結合,讓GFP的熒光增強。


      這項研究表明,納米抗體能夠在體外和體內操控蛋白的構象。Rothbauer還表示,他們正在研究用于高分辨率顯微鏡的GFP特異的納米抗體。他們也在篩選其它熒光蛋白的納米抗體。因此,在不遠的將來,生物學家應該有越來越多的工具可供選擇。


      參考文獻

      Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2009).


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