Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries
Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries
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DOI：
10.1073/pnas.1013910107
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发表时间：
2010-11-23
影响因子：
11.1
通讯作者：
Mayo, Stephen L.
中科院分区：
文献类型：
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作者：
Chica, Roberto A.;Moore, Matthew M.;Mayo, Stephen L.
红色荧光蛋白(RFP)发射波长较长，对全动物成像具有吸引力，因为细胞对红光更透明。虽然已经使用定向进化开发了几个有用的RFP，但对进一步红移和改进RFP的探索仍在继续。在此，我们报道了一种基于结构的合理设计方法来红移RFP的荧光发射。我们应用了一种计算和实验相结合的方法，使用计算蛋白质设计作为电子预筛选来生成mCherry突变体的有针对性的组合库。计算程序帮助我们确定了可以满足相互作用的残基，这些相互作用假设会导致红移，而不会破坏蛋白质折叠的稳定。这些相互作用包括通过与酰亚胺氧原子的氢键来稳定激发态，通过疏水堆积在带电的苯酚周围而使基态不稳定，以及通过pi堆积作用来稳定激发态。我们的方法使我们能够鉴定出三个mCherry突变体(mRojoA、mRojoB和mRouge)，它们显示发射波长>；630 nm，代表20-26 nm的红移。此外，我们的方法需要对总共5000个类似克隆进行实验筛选，这个数字比以前用来实现可比红移的克隆要小几个数量级。此外，mRojoA和mRouge的晶体结构允许我们验证假设的导致红移的相互作用的实现，支持它们对观察到的红移的贡献。
The longer emission wavelengths of red fluorescent proteins (RFPs) make them attractive for whole-animal imaging because cells are more transparent to red light. Although several useful RFPs have been developed using directed evolution, the quest for further red-shifted and improved RFPs continues. Herein, we report a structure-based rational design approach to red-shift the fluorescence emission of RFPs. We applied a combined computational and experimental approach that uses computational protein design as an in silico prescreen to generate focused combinatorial libraries of mCherry mutants. The computational procedure helped us identify residues that could fulfill interactions hypothesized to cause red-shifts without destabilizing the protein fold. These interactions include stabilization of the excited state through H-bonding to the acylimine oxygen atom, destabilization of the ground state by hydrophobic packing around the charged phenolate, and stabilization of the excited state by a pi-stacking interaction. Our methodology allowed us to identify three mCherry mutants (mRojoA, mRojoB, and mRouge) that display emission wavelengths >630 nm, representing red-shifts of 20-26 nm. Moreover, our approach required the experimental screening of a total of similar to 5,000 clones, a number several orders of magnitude smaller than those previously used to achieve comparable red-shifts. Additionally, crystal structures of mRojoA and mRouge allowed us to verify fulfillment of the interactions hypothesized to cause red-shifts, supporting their contribution to the observed red-shifts.