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Bioquímica

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Published: May 30, 2021
doi:
10.3791/62655
,
1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus,The Hebrew University of Jerusalem, 2The Center for Nanoscience and Nanotechnology,The Hebrew University of Jerusalem

Summary

时间分辨单分子蛋白诱导的荧光增强是一种有用的荧光光谱接近传感器，对蛋白质的局部结构变化敏感。在这里，我们展示了它可用于揭示α-突触核蛋白中稳定的局部构象，当使用较长距离的FRET标尺测量时，这也称为球状非结构化和不稳定。

Abstract

使用光谱标尺来跟踪单个生物分子及其动力学的多种构象，彻底改变了对结构动力学及其对生物学的贡献的理解。虽然基于FRET的标尺报告了3-10nm范围内的染料间距离，但其他光谱技术，如蛋白质诱导荧光增强（PIFE），报告了染料与蛋白质表面在较短的0-3nm范围内的接近度。无论选择哪种方法，它在测量自由扩散的生物分子时的使用一次一个检索实验参数的直方图，产生单独的中心分布的生物分子亚群，其中每个亚群代表在毫秒内保持不变的单个构象，或者相互转换速度远快于毫秒的多个构象，因此平均输出子群。在单分子FRET中，直方图中报告的参数是染料间FRET效率，一种内在无序的蛋白质，例如缓冲液中的α-突触核蛋白单体，先前被报道为表现出多个构象的单个平均亚群快速相互转化。虽然这些过去的发现取决于基于FRET的标尺的3-10nm范围，但我们试图使用单分子PIFE对这种蛋白质进行测试，在那里我们跟踪位点特异性sCy3标记的α突触核蛋白的荧光寿命一次一个。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具有明显不同平均寿命的寿命亚群，在10-100 ms内相互转换。这些结果表明，虽然α-突触核蛋白可能在全球范围内紊乱，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光谱接近传感器的优势，这些传感器一次跟踪一个生物分子的局部或全局结构变化。

Introduction

在过去的二十年中，基于单分子荧光的方法已成为测量生物分子1，2的强大工具，探索不同的生物分子参数如何分布以及它们如何在亚毫秒分辨率3，4，5下动态地在这些参数的不同亚群之间相互转换。这些技术中的参数包括FRET测量的能量传递效率6，7，荧光各向异性8，9，荧光量子产率和寿命10，11，作为不同荧光猝灭12或增强13机制的函数。其中一种机制，更广为人知的是蛋白质诱导荧光增强（PIFE）14，引入了荧光量子产率和寿命的增强，作为激发态时荧光团自由异构化的空间位阻的函数，由染料附近的蛋白质表面引起14，15，16，17，18，19.FRET和PIFE都被认为是光谱尺或接近传感器，因为它们的测量参数与被测量的标记生物分子内的空间测量直接相关。虽然FRET效率与3-10 nm20范围内一对染料之间的距离有关，但PIFE跟踪荧光量子产量的增加或寿命与染料与附近蛋白质表面之间的距离在0-3 nm19范围内有关。

单分子FRET已被广泛用于为许多不同的蛋白质系统提供结构见解，包括内在无序蛋白质（IDPs）21，如α-突触核蛋白（α-Syn）22。α-Syn在与不同的生物分子结合后，在不同的条件下可以形成有序的结构23，24，25，26，27，28，29，30。然而，当未结合时，α-Syn单体的特征在于具有高构象异质性，具有快速相互转化的构象31，32。

α-Syn的构象以前已经使用各种不同的技术进行了研究，这些技术有助于识别这种高度异质和动态的蛋白质系统的构象动力学33，34，35，36，37，38，39。有趣的是，缓冲液中α-Syn的单分子FRET（smFRET）测量报告了单个FRET群体39，40，这是构象的时间平均动态相互转换的结果，其时间比α-Syn通过共聚焦点的典型扩散时间快得多（相对于典型的毫秒扩散时间，时间快到几微秒，甚至比这更快）40， 41.然而，使用具有3-10nm距离灵敏度的FRET光谱标尺有时只能报告小蛋白质（如α-Syn）的整体结构变化。利用具有较短距离灵敏度的光谱接近传感器进行单分子测量有可能报告局部结构的动力学。在这里，我们对α-Syn进行单分子PIFE测量，并确定荧光寿命的不同亚群映射到不同的局部结构，它们之间的过渡速度慢至100 ms。这项工作总结了自由扩散的α-Syn分子的时间分辨smPIFE测量值，一次一个，在缓冲液中，以及当与基于SDS的膜结合时作为短程单分子光谱接近传感器。

Protocol

1. 质粒转化 制备0.5 L SOC培养基 称取10克色蛋白酮，2.5克酵母提取物，0.25克氯化钠（NaCl），0.1克氯化钾（KCl）。 加入双蒸馏水（DDW），直至总体积为0.5升。 通过加入氢氧化钠（NaOH）调节至pH 7。 准备100 mL的配分试样和高压灭菌器。 使用前，将0.5毫升无菌氯化镁（MgCl2）和1.8毫升无菌葡萄糖加入100毫升SOC中。 在冰上解冻BL21（…

Representative Results

作为IDP，当它不与另一种生物分子结合时，α-Syn表现出多种构象之间的结构动力学，在几微秒40甚至数百纳秒41下过渡 。当α-Syn穿过共聚焦点时，它可能会在构象之间经历数千次转变。事实上，当smFRET使用39，40时就是这种情况。在这里，我们进行smPIFE测量，以探测α-Syn的局部构象动力学。 测量记…

Discussion

进行了广泛的生化和生物物理研究，以研究α-Syn的结构特征及其无序性质33，34，35，36，37，38。一些工作已经利用自由扩散的smFRET来研究无结合的α-Syn单体的分子内动力学。这些工作报告了α-Syn的高动态异质性，这导致在通过共聚焦点的典型扩散时…

Declarações

The authors have nothing to disclose.

Acknowledgements

编码A56C α-Syn突变体的pT-t7质粒是作为Asaf Grupi博士，Dan Amir博士和Elisha Haas博士的礼物送给我们的。本文得到了美国国立卫生研究院（NIH，授予R01 GM130942给E.L.作为子奖），以色列科学基金会（KillCorona – 遏制冠状病毒研究计划中的3565/20赠款），米尔纳基金和耶路撒冷希伯来大学（启动基金）的支持。

Materials

Amicon Ultra-15 Centrifugal Filter Units Merc C7715 cutoff: 100 kDa
ammonium sulfate Sigma-Aldrich A4418
BSA Sigma-Aldrich A9647
cysteamine Sigma-Aldrich 30070
dialysis bags – MEGA GeBaFlex-tube Gene Bio-Application MEGA320
dithiothreitol (DTT) Sigma-Aldrich 43815
ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich E5134
Fast SeeBand staining solution Gene Bio-Application SB050
Glycine Sigma-Aldrich 50046
D-Glucose Sigma-Aldrich G7021
HEPES Sigma-Aldrich 54457
HiTrap Desalting 5 mL Sigma-Aldrich GE17-1408
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX) Sigma-Aldrich 238813
isopropyl β-d-1-thiogalactopyranoside (IPTG) Sigma-Aldrich I5502
LB broth Sigma-Aldrich L3152
Magnesium chloride Sigma-Aldrich 63068
MonoQ column Sigma-Aldrich 54807
protein LoBind tube Sigma-Aldrich EP0030108094 0.5 mL
Rinse a µ-slide 18 Ibidi 81816
SDS Sigma-Aldrich 75746
Sodium acetate Sigma-Aldrich S2889
Sodium hydroxide Sigma-Aldrich S8045
Sodium phosphate monobasic monohydrate Sigma-Aldrich 71507
Sterile Cell spreaders, Drigalski spatulas mini-plast 815-004-05-001
streptomycin sulfate Sigma-Aldrich S9137
sulfo-Cy3 maleimide abcam ab146493
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) Sigma-Aldrich 75259
Tris-HCl Sigma-Aldrich 93363
Tryptone Sigma-Aldrich T7293
Yeast Extract Sigma-Aldrich Y1625
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Tags

Time-resolvedProtein-induced Fluorescence EnhancementSingle-moleculeAlpha-synucleinStructural SubpopulationsLocal ConformationsSulfo-Cy3BSAWater Immersion ObjectiveLaser FocusPhoton DetectionFRETBurstsPhoton-HDF5

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Zaer, S., Lerner, E. Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time. J. Vis. Exp. (171), e62655, doi:10.3791/62655 (2021).
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