Department of Chemistry , University of Michigan , Ann Arbor , Michigan 48109 , United States.
Department of Chemistry, Biology and Biotechnology , University of Perugia , Perugia 06123 , Italy.
J Am Chem Soc. 2018 Nov 21;140(46):15731-15743. doi: 10.1021/jacs.8b08208. Epub 2018 Nov 9.
In this investigation, we report evidence for energy transfer in new protein-based megamolecules with tunable distances between donor and acceptor fluorescent proteins. The megamolecules used in this work are monodisperse oligomers, with molecular weights of ∼100-300 kDa and lengths of ∼5-20 nm, and are precisely defined structures of fusion protein building blocks and covalent cross-linkers. Such structures are promising because the study of energy transfer in protein complexes is usually difficult in this long length regime due to synthetic limitations. We incorporated fluorescent proteins into the megamolecule structure and varied the separation distance between donor and acceptor by changing the length of the cross-linker in dimer conjugates and inserting nonfluorescent spacer proteins to create oligomers. Two-photon absorption measurements demonstrated strong coupling between donor and acceptor dipoles in the megamolecules. For the dimer systems, no effect of the cross-linker length on energy transfer efficiency was observed with the steady-state fluorescence investigation. However, for the same dimer conjugates, energy transfer rates decreased upon increasing cross-linker length, as evaluated by fluorescence up-conversion. Molecular dynamics simulations were used to rationalize the results, providing quantitative agreement between measured and calculated energy transfer lengths for steady-state results, and showing that the differences between the time-resolved and steady-state measurements arise from the long time scale for large-scale fluctuations in the megamolecule structure. Our results show an increase in energy transfer length with increasing megamolecule size. This is evidence for long-range energy transfer in large protein megamolecules.
在这项研究中，我们报告了在新的基于蛋白质的巨型分子中存在能量转移的证据，这些巨型分子的供体和受体荧光蛋白之间的距离可以调节。在这项工作中使用的巨型分子是单分散的低聚物，分子量约为 100-300 kDa，长度约为 5-20nm，是融合蛋白构建块和共价交联剂的精确定义结构。由于合成限制，在这个长链长度范围内，蛋白质复合物中的能量转移研究通常很困难，因此这种结构很有前景。我们将荧光蛋白掺入巨型分子结构中，并通过改变二聚体缀合物中交联剂的长度和插入非荧光间隔蛋白来改变供体和受体之间的分离距离，从而创建低聚物。双光子吸收测量表明，在巨型分子中供体和受体偶极子之间存在强耦合。对于二聚体系统，稳态荧光研究没有观察到交联剂长度对能量转移效率的影响。然而，对于相同的二聚体缀合物，通过荧光上转换评估，随着交联剂长度的增加，能量转移速率降低。分子动力学模拟用于合理化结果，为稳态结果提供了测量和计算能量转移长度之间的定量一致性，并表明时间分辨和稳态测量之间的差异源于巨型分子结构中大规模波动的长时间尺度。我们的结果表明，随着巨型分子尺寸的增加，能量转移长度增加。这证明了在大型蛋白质巨型分子中存在长程能量转移。
