Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Blc Tags for Protein-PAINT Super-resolution Microscopy.,ACS Chemical Biology

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Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Blc Tags for Protein-PAINT Super-resolution Microscopy.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-08-18 , DOI: 10.1021/acschembio.0c00440
Liya Muslinkina ₁ , Alexey S Gavrikov ₂ , Nina G Bozhanova ₃ , Alexander S Mishin ₂ , Mikhail S Baranov ₂ , Jens Meiler _{3,

4} , Nadya V Pletneva ₂ , Vladimir Z Pletnev ₂ , Sergei Pletnev ₁

Affiliation

Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.

中文翻译：

用于蛋白质-PAINT超分辨率显微镜的两个增强型细菌脂蛋白Blc标签的基于结构的合理设计。

活细胞中的超分辨率荧光成像在技术上仍然具有挑战性，这主要归因于荧光标签的光分解。最近提出的蛋白质-PAINT是唯一可用于对活细胞中蛋白质进行长时间成像的超分辨率技术。它是通过表达为融合物的氟激活蛋白和溶剂变色合成染料的复合物来实现的。光漂白后，将络合物中的染料替换为样品中可用的新鲜氟。通过适当的动力学，这种替代可产生获得超分辨率所需的荧光闪烁，并克服了与不可替代的荧光团损失相关的光致漂白。在这里，我们报告了基于DiB1：M739复合物的1.58Å晶体结构的两个蛋白质PAINT标签的合理设计，改进的绿色发光DiB3 / F74V：M739和新的橙色发光DiB3 / F53L：M739。他们超越了以前报道的基于DiB的标签，成为蛋白质PAINT的同类最佳生物标志物。新标签将蛋白质PAINT从概念验证发展为一种可靠的工具，适用于对固定和活细胞中的细胞内蛋白质进行长时间的超分辨率成像，以及使用单一荧光素的两种颜色的PAINT类纳米检查。

更新日期：2020-09-20

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