Fraunhofer EMFT, Hansastr. 27 D, München, Germany.
Phys Chem Chem Phys. 2011 Oct 21;13(39):17852-63. doi: 10.1039/c1cp21088b. Epub 2011 Sep 5.
This article reports the full characterisation of the optical properties of a biosynthesised protein consisting of fused cyan fluorescent protein, glucose binding protein and yellow fluorescent protein. The cyan and yellow fluorescent proteins act as donors and acceptors for intramolecular fluorescence resonance energy transfer. Absorption, fluorescence, excitation and fluorescence decays of the compound protein were measured and compared with those of free fluorescent proteins. Signatures of energy transfer were identified in the spectral intensities and fluorescence decays. A model describing the fluorescence properties including energy transfer in terms of rate equations is presented and all relevant parameters are extracted from the measurements. The compound protein changes conformation on binding with calcium ions. This is reflected in a change of energy transfer efficiency between the fluorescent proteins. We track the conformational change and the kinetics of the calcium binding reaction from fluorescence intensity and decay measurements and interpret the results in light of the rate equation model. This visualisation of change in protein conformation has the potential to serve as an analytical tool in the study of protein structure changes in real time, in the development of biosensor proteins and in characterizing protein-drug interactions.
本文报道了一种由融合的青色荧光蛋白、葡萄糖结合蛋白和黄色荧光蛋白组成的生物合成蛋白质的光学性质的全面特征。青色和黄色荧光蛋白作为分子内荧光共振能量转移的供体和受体。测量了该复合蛋白的吸收、荧光、激发和荧光衰减，并与游离荧光蛋白的测量值进行了比较。在光谱强度和荧光衰减中鉴定出了能量转移的特征。提出了一个用速率方程描述荧光性质包括能量转移的模型，并从测量中提取了所有相关参数。该复合蛋白在与钙离子结合时会发生构象变化。这反映在荧光蛋白之间的能量转移效率的变化上。我们从荧光强度和衰减测量中跟踪构象变化和钙结合反应的动力学，并根据速率方程模型解释结果。这种蛋白质构象变化的可视化有可能成为实时研究蛋白质结构变化、开发生物传感器蛋白和表征蛋白质-药物相互作用的分析工具。
