Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time.
Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time.
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DOI：
10.3791/62655
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发表时间：
2021-05-30
期刊：
影响因子：
--
通讯作者：
Lerner E
中科院分区：
文献类型：
--
作者：
Zaer S;Lerner E
使用光谱尺追踪单个生物分子的多种构象及其动力学，彻底改变了对结构动力学及其对生物学贡献的理解。基于 FRET 的标尺报告 3-10 nm 范围内的染料间距离，而其他光谱技术（例如蛋白质诱导荧光增强 (PIFE)）则报告较短的 0-3 nm 范围内的染料与蛋白质表面之间的接近度。无论选择哪种方法，它在测量自由扩散的生物分子时一次检索一个实验参数的直方图，产生单独的集中分布的生物分子子群，其中每个子群要么代表在毫秒内保持不变的单个构象，要么代表比毫秒快得多的相互转换的多个构象，因此是一个平均的子群。在单分子 FRET 中，直方图中报告的参数是染料间 FRET 效率，一种本质上无序的蛋白质，例如缓冲液中的 α-突触核蛋白单体，之前被报道为表现出快速相互转换的多个构象的单个平均子群。虽然这些过去的发现取决于基于 FRET 的标尺的 3-10 nm 范围，但我们试图使用单分子 PIFE 测试这种蛋白质，我们一次跟踪一个位点特异性 sCy3 标记的 α-突触核蛋白的荧光寿命。有趣的是，使用这种较短距离的光谱接近传感器，sCy3 标记的 α-突触核蛋白表现出几个平均寿命明显不同的寿命亚群，这些亚群在 10-100 毫秒内相互转换。这些结果表明，虽然 α-突触核蛋白可能在整体上无序，但它仍然获得了稳定的局部结构。总之，在这项工作中，我们强调了使用不同的光谱接近传感器一次跟踪一个生物分子的局部或全局结构变化的优势。
Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3–10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0–3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the α-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3–10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled α-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled α-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10–100 ms. These results show that while α-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.
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影响因子：
3.7
作者：
Ingargiola A;Lerner E;Chung S;Panzeri F;Gulinatti A;Rech I;Ghioni M;Weiss S;Michalet X
通讯作者：
Michalet X
影响因子：
4.3
作者：
Brodie, Nicholas I.;Popov, Konstantin I.;Borchers, Christoph H.
通讯作者：
Borchers, Christoph H.
DOI：
10.1073/pnas.95.4.1556
发表时间：
1998-02-17
影响因子：
11.1
作者：
Eggeling, C;Fries, JR;Seidel, CAM
通讯作者：
Seidel, CAM
DOI：
10.1073/pnas.090104997
发表时间：
2000-05-09
影响因子：
11.1
作者：
Deniz, AA;Laurence, TA;Weiss, S
通讯作者：
Weiss, S
影响因子：
2.9
作者：
Fries, JR;Brand, L;Seidel, CAM
通讯作者：
Seidel, CAM