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PNAS:科学家发现可开关型的绿色荧光蛋白

来源:华文生技网 2007-04-19 09:14

     奥勒岗大学(University of Oregon)的科学家发现绿色荧光蛋白(green fluorescent proteins),在置入一个单氧原子后,能持续维持不发光的状态达65小时之久,这项研究结果发表于近期的PNAS期刊。S. James Remington教授表示:「能控制荧光蛋白的荧光开关,将使荧光蛋白在分子生物学的应用上更加广泛。」

    荧光蛋白最初从水母中被分离出来,后来在珊瑚礁中发现许多不同颜色的荧光蛋白,将分子生物学的实验彻底革新。研究人员利用这些荧光蛋白作为基因表现与否的标志,能追踪、观察细胞内蛋白质活性的表现。相较于荧光蛋白,可开关型的荧光蛋白(Photoswitchable fluorescent proteins)有许多优势,Remington教授说:你可以将所有的分子都标记上这些可开关型的荧光蛋白,在显微镜下利用雷射光仅活化一部分的分子,依实验的设计来开或关你要观察的分子。

    UO的博士班学生J. Nathan Henderson解开了来自于海葵的高分辨率荧光蛋白质结构,包括了发光与不发光状态等两种结构。在发光的情况下,两条支链的原子以共面的型态(coplanar fashion)整齐排列,当受到雷射光照射时,支链原子的角度也跟着改变,形成非共面且不稳定的排列,荧光蛋白很迅速的变成不发光的状态,这两种不同的结构让研究人员有机会观察到邻近原子的互动情况。

    Remington教授说:「荧光蛋白在不发光的状态下,其分子会吸收紫外光(Ultraviolet light)而不会发射出任何光线,然而,当发色基团(chromophore)吸收紫外光时,偶而会被离子化而使分子形成负价电荷,而导致分子转变成发荧光状态。」Henderson在研究中发现,荧光蛋白在不发光的阶段,结构中的碳与氧原子是不利于交互作用的,他在蛋白结构中适当的地方置入氧原子,没想到这个突变的荧光蛋白竟然能在 “开” 与 ”关” 的延迟时间从原本的5分钟延长到65小时之久。Remington教授说:「能控制荧光蛋白的开与关将能促使光学记忆的改善,例如:单一分子信息的贮存,此外,还能强化荧光显微镜的功能以及分子标志的作业等应用。」

     (资料来源 : Bio.com)

部分英文原文:

Published online before print April 9, 2007, 10.1073/pnas.0700059104
PNAS | April 17, 2007 | vol. 104 | no. 16 | 6672-6677


Structural basis for reversible photobleaching of a green fluorescent protein homologue

J. Nathan Henderson,, Hui-wang Ai, Robert E. Campbell, and S. James Remington,¶,||

Departments of Chemistry and ¶Physics, and Institute of Molecular Biology, University of Oregon, Eugene, OR 97403; and Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2

Edited by Martin Chalfie, Columbia University, New York, NY, and approved February 28, 2007 (received for review January 3, 2007)

Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cis–trans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.

Fig. 2. Stereoviews showing electron density and superimposed atomic
models in the A monomers of mTFP0.7. Water molecules are represented as
red spheres labeledWorW*. The chromophores are labeled Chrom. (A) 2Fo
Fc maps (green) for the fluorescent-state model contoured at 1.8 . (B) The
mTFP0.7 fluorescent-state model chromophore environment emphasizing
hydrogen-bond partners and the quadrupole salt-bridge network adjacent to
the chromophore. Hydrogen bonds are represented with black dashed lines
(distances are given in angstroms). (C) 2Fo  Fc maps (green) for the photobleached
dark state model, contour level 1.0 . (D) The chromophore environment
of the photobleached state model (apparent close contacts are
shown in red

 

 

crystallography | fluorescence | photoswitching | protein structure

Author contributions: J.N.H. and S.J.R. designed research; J.N.H. performed research; H.-w.A. and R.E.C. contributed new reagents/analytic tools; J.N.H. analyzed data; and J.N.H., H.-w.A., R.E.C., and S.J.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2OTB and 2OTE).

This article contains supporting information online at www.pnas.org/cgi/content/full/0700059104/DC1.

||To whom correspondence should be addressed. E-mail: jremington@uoxray.uoregon.edu

英文全文链接:www.pnas.org/cgi/content/full/104/16/6672

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