在细胞生物学研究中，荧光蛋白几乎无处不在，因此科学家对它的改造也在层层升级。eGFP应该是大家最熟悉的一种增强型变异体，与野生型的GFP相比，亮度更强，光稳定性更好。然而，科学的探索是永无止境的，研究人员还在寻觅更好的荧光蛋白。这不，德国慕尼黑大学的Ulrich Rothbauer和他的同事就另辟蹊径，采用一种独特的方法来改变GFP的特性。文章发表在近期的《Nature Structural & Molecular Biology》上。

他们的秘密武器是纳米抗体（nanobody）。这些来源于骆驼科动物的单结构域肽段与抗体相似，都有着与抗原结合的性质，不过它们的稳定性更好，体积更小。根据这一性质，研究人员尝试筛选出能调节GFP构象及光谱性质的纳米抗体。利用噬菌体展示筛选，他们鉴定出7种能与GFP结合的不同纳米抗体。

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.

其中一个纳米抗体与GFP结合后，荧光比eGFP本身增强了4倍，后来他们将之命名为Enhancer。结构分析表明，在GFP-Enhancer的复合体中，发光基团带有负电荷。而Enhancer与eGFP的结合只让荧光增强了1.5倍，于是作者们推测Enhancer的结合行使着与eGFP的突变类似的功能，从而提高了荧光强度。实际上，Enhancer-GFP的吸收光谱与eGFP相似。

同时，研究人员还发现了另一个纳米抗体，在结合后能将GFP的荧光强度降低5倍，他们称其为Minimizer。

Structures of the GFP–nanobody complexes. (a–d) Enhancer (a; light blue ribbon model) and Minimizer (c) (orange ribbon model) recognize two different nonlinear epitopes on the surface of the GFP â-can (green ribbon model). The insets in a and c 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的调节不仅发生在体外，在活细胞内也能重演。作者们将Enhancer用于一系列体内应用中，包括带GFP标签的雌激素受体在Hela细胞中的运输。在激素诱导下，重组子转位到细胞核，在那里它与核定位的Enhancer结合，让GFP的荧光增强。

这项研究表明，纳米抗体能够在体外和体内操控蛋白的构象。Rothbauer还表示，他们正在研究用于高分辨率显微镜的GFP特异的纳米抗体。他们也在筛选其它荧光蛋白的纳米抗体。因此，在不远的将来，生物学家应该有越来越多的工具可供选择。

参考文献

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