Complex System Research Division, Beijing Computational Science Research Center, Beijing, China.
Key Lab of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu, China.
Proteins. 2019 Jul;87(7):531-540. doi: 10.1002/prot.25676. Epub 2019 Feb 27.
Green fluorescent protein (GFP) is a widely used biomarker that demands systematical rational approaches to its structure function redesign. In this work, we mainly utilized atomistic molecular dynamics simulations to inspect and visualize internal fluctuation and coordination around chromophore inside GFP, from water to nonpolar octane solvent. We found that GFP not only maintains its β-barrel structure well into the octane, but also sustains internal residue and water coordination to position the chromophore stably while suppress dihedral fluctuations of the chromophore, so that functional robustness of GFP is achieved. Our accompanied fluorescence microscope measurements accordingly confirmed the GFP functioning into the octane. Furthermore, we identified that crucial water sites inside GFP along with permeable pores on the β-barrel of the protein are largely preserved from the water to the octane solvent, which allows sufficiently fast exchanges of internal water with the bulk or with the water layer kept on the surface of the protein. By additionally pulling GFP from bulk water to octane, we suggest that the GFP function can be well maintained into the nonpolar solvent as long as, first, the protein does not denature in the nonpolar solvent nor across the polar-nonpolar solvent interface; second, a minimal set of water molecules are in accompany with the protein; third, the nonpolar solvent molecules may need to be large enough to be nonpermeable via the water pores on the β-barrel.
绿色荧光蛋白（GFP）是一种广泛应用的生物标志物，需要系统的理性方法来对其结构和功能进行重新设计。在这项工作中，我们主要利用原子分子动力学模拟来检查和可视化 GFP 中发色团内部的内部波动和配位情况，从水到非极性辛烷溶剂。我们发现，GFP 不仅在辛烷中很好地保持其β-桶结构，而且在稳定发色团位置的同时保持内部残基和水分子的配位，抑制发色团的二面角波动，从而实现 GFP 的功能稳健性。我们相应的荧光显微镜测量结果证实 GFP 在辛烷中仍能发挥作用。此外，我们确定 GFP 内部的关键水分子以及蛋白质β-桶上的可渗透孔在从水到辛烷溶剂的过程中都得到了很大程度的保留，这允许内部水分子与主体或保留在蛋白质表面的水分子层之间进行足够快速的交换。通过将 GFP 从主体水中进一步拉到辛烷中，我们提出只要满足以下三个条件，GFP 的功能就能很好地在非极性溶剂中保持：第一，蛋白质在非极性溶剂中不会变性，也不会在极性-非极性溶剂界面变性；第二，蛋白质有一小部分水分子伴随；第三，非极性溶剂分子需要足够大，不能通过β-桶上的水分子孔渗透。
