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Biochimica

福斯特共振能量转移映射：阐明全球结构特征的新方法

Published: March 16, 2022
doi:
10.3791/63433
, ,
1Molecular Biology and Biochemistry Department,Wesleyan University, 2Molecular Biophysics Program,Wesleyan University

Summary

该研究详细介绍了FRET映射的方法，包括标记位点的选择，染料的选择，采集和数据分析。该方法可有效确定蛋白质系统中的结合位点、构象变化和动态运动，如果与现有的3-D结构信息结合使用，这种方法最有用。

Abstract

Förster共振能量转移（FRET）是一种基于荧光的成熟方法，用于成功测量 体外 生物分子内和细胞内的距离。在FRET中，通过荧光强度或寿命的变化来测量能量转移的效率与两个荧光分子或标记之间的距离有关。从远处测定动力学和构象变化只是该方法应用于生物系统的一些示例。在某些条件下，该方法可以通过提供有关动力学，柔韧性和对结合表面的适应性的信息来增加和增强现有的X射线晶体结构。我们描述了使用FRET和相关距离测定来阐明结构性质，通过识别结合位点或二聚体亚基的方向。通过明智地选择标记位点，并经常采用多种标记策略，我们成功地应用了这些映射方法来确定蛋白质-DNA复合物和SecA-SecYEG蛋白质易位系统中的全局结构性质。在SecA-SecYEG系统中，我们使用FRET映射方法来识别前蛋白结合位点并确定结合信号序列区域的局部构象。本研究概述了进行FRET绘图研究的步骤，包括识别适当的标记位点，讨论可能的标记，包括非天然氨基酸残基，标记程序，如何进行测量以及解释数据。

Introduction

对于蛋白质，动力学的阐明以及3维（3-D）结构知识可以增强对生物分子系统的结构 – 功能关系的理解。结构方法，如X射线晶体学和低温电子显微镜，捕获静态结构，并且通常需要确定多个结构以阐明生物分子结合和动力学的各个方面1。本文讨论了一种基于解决方案的方法来映射全局结构元素，例如结合位点或结合相互作用，这些元素可能更短暂，并且不容易被静态方法捕获。这种方法的强候选系统是先前已通过X射线晶体学，NMR光谱或其他结构方法确定3-D结构的系统。在这种情况下，我们利用SecA-SecYEG复合物的X射线晶体结构（蛋白质一般分泌途径中的核心参与者），在前蛋白穿过膜2之前，使用Förster共振能量转移（FRET）绘制信号肽结合位点的位置。通过基因修饰对生物系统的操纵加上我们对3-D结构的了解，使得能够在插入通道 3之前立即确定信号序列和早期成熟区域的构象。

FRET涉及能量从一个分子（供体）到另一个分子（受体）的无辐射转移，其距离依赖性方式是通过空间4，5。通过供体的减少或受体荧光强度的增加来监测这种转移的效率。能量传递的效率可谓

E = R06/（R06 + R6）

其中 R0 值是传输效率为 50% 的距离6。该技术以前被描述为分子尺，并且根据供体 – 受体染料4，7，8，9的身份，有效地确定2.5-12nm范围内的距离。使用或不使用受体的供体荧光强度和寿命允许确定转移效率，因此距离为5，8。由于该技术的可用性，该方法的灵敏度和易用性，FRET在单分子荧光光谱和共聚焦显微镜6等领域也得到了广泛的应用。荧光蛋白（如绿色荧光蛋白）的出现使得细胞内动力学和活细胞成像的观察相对容易10，11。许多 FRET 应用程序（如此类）将在此虚拟问题中详细讨论。

在这项研究中，我们特别关注使用FRET测量来产生距离值以确定结构细节。以前，FRET测量已被有效地用于确定DNA分子在与蛋白质12，13，14结合时的构象，蛋白质的内部动力学以及蛋白质结合相互作用15，16，17。这种方法的优点在于能够确定具有相对较少材料量的溶液中的柔性和动态结构单元。值得注意的是，当与现有结构信息结合使用时，这种方法特别有效，不能用作3-D结构测定的手段。如果工作建立在现有的结构信息之上，并且通常与计算仿真18，19相结合，则该方法可提供最佳的结构洞察力和细化。在这里，描述了使用从稳态和时间分辨的FRET测量中获得的距离来绘制结合位点，其位置尚不清楚，在SecA-SecYEG复合物的现有晶体结构上，主要蛋白质在一般分泌途径3中。

一般分泌途径是一种高度保守的系统，从原核生物到真核生物再到古菌，介导蛋白质穿过或进入膜到其在细胞中的功能位置的运输。对于革兰氏阴性菌，例如我们研究中使用的生物体 大肠杆菌 ，蛋白质入或穿过内膜转移到周质。细菌SecY通道复合物（称为转座蛋白）与其他蛋白质协调以转移新合成的蛋白质，该蛋白质通过通常位于N端20，21的信号序列被引导到其在细胞中的正确位置。对于与围质结合的蛋白质，ATPase SecA蛋白与核糖体的出口隧道相关，并且在翻译了大约100个残基22个后与前蛋白相关。与SecB伴侣蛋白一起，它将前蛋白维持在未折叠状态。SecA与SecYEG转座结合，并通过ATP水解的许多循环，促进蛋白质在膜上的转运23，24。

SecA是一种多结构域蛋白，以胞质和膜结合形式存在。SecA是细胞质基质中的同源二甲基蛋白，由前蛋白结合或交联结构域25，两个核苷酸结合结构域，螺旋翼结构域，螺旋支架结构域和两个螺旋指（THF）26，27，28，29 组成（图1）。在先前对SecA-SecYEG复合物的晶体学研究中，THF的位置表明它积极参与蛋白质易位，随后与信号肽的交联实验进一步确定了该区域在蛋白质易位30，31中的重要性。先前使用FRET映射方法的研究表明，外源信号肽与SecA2，32的该区域结合。为了在插入SecYEG通道之前充分了解信号序列的构象和位置以及前蛋白的早期成熟区域，创建了一种蛋白质嵌合体，其中通过Ser-Gly连接子将早期成熟区域的信号序列和残基连接到SecA（图1）。使用这种生物学上可行的结构，进一步证明前蛋白的信号序列和早期成熟区域以平行的方式与THF结合2。随后，使用FRET映射方法阐明了在SecYEG存在下信号序列和早期成熟区域的构象和位置，如下所述3。

了解SecA-SecYEG复合物33，34，35的3-D结构和结合位点的可能位置，使我们能够明智地将供体 – 受体标签放置在各个FRET距离的交集识别结合位点位置的位置。这些FRET映射测量表明，信号序列和前蛋白的早期成熟区域形成发夹，尖端位于SecYEG通道的口，表明发夹结构在通道插入之前是模板化的。

Protocol

1. 标签站点的选择 鉴定至少三个潜在的标记位点，以对现有蛋白质结构上的假定结合位点进行三角测量。在这种情况下，鉴定出通过遗传融合附着在SecA上的SecA，SecYEG和前蛋白2。 选择在推定结合位点的25-75 Å范围内的标记位点，并且在蛋白质的相对静态区域中，距离将决定要使用的特定FRET染料对36。将标记位点定位在彼此相对不同的?…

Representative Results

这项研究的重点是在将前蛋白插入SecYEG通道之前确定SecA上前蛋白结合位点的位置。为了绘制结合位点，在前蛋白的不同区域和SecA和SecYEG蛋白上的三个不同位置之间进行了FRET实验（图1A-D）。从获得的距离和SecA，SecYEG和前蛋白的三维结构中，预测了前蛋白结合位点的位置。不是使用三个独立的实体（SecA，SecYEG和前蛋白）来执行这些测量，而是在掺入…

Discussion

通过使用FRET映射方法，我们鉴定了SecA蛋白上的信号序列结合位点。重要的是，该复合物的3-D晶体结构的存在极大地促进了我们的研究。这种制图方法的优势在于能够使用现有结构来识别要标注的位置。这种方法不能用于确定3D结构;然而，结构元件56的测定、现有结构49的细化、结合位点位置2、32的测定或动态运动<sup …

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院拨款R15GM135904（授予IM）和美国国立卫生研究院拨款GM110552（授予DBO）的支持。

Materials

490 nm LED laser Horiba 1684-LED
Alexa Fluor 647 C2 Maleimide//DIBO Alkyne Life Technologies A20347
Agar Difco DF0812
Alexa Fluor 488 C5 Maleimide/DIBO Alkyne Life Technologies A10254
Alexa Fluor 488 DIBO Alkyne Life Technologies S10904
Alexa Fluor 647 DIBO Alkyne Life Technologies S10906
Amicon Ultra­4 Centrifugal filter (50kDa MWCO) Sigma UFC805008
Dodecylmaltoside (DDM) Anatrace D310
E. coli alkaline phosphatase signal peptide SP22 Biomolecules Midwest N/A Synthesized custom item
extended signal peptide SP41 Biomolecules Midwest N/A Synthesized custom item
FluorEssence Horiba version 2.4 spectral acquisition program for Fluoromax4 spectrofluorometer
Fluoromax 4 spectrofluorometer Horiba N/A
GlobalsWE Laboratory for Fluorescence Dynamics, University of California, Irvine spectral analysis program for time-resolved decays
H­4­Azido­Phe­OH BACHEM 4020250.0001
LB (Miller) Broth Fisher Scientific BP9723
Ludox HS-40 colloidal silica (40 wt.% suspension in H2O) Sigma-Aldrich 420816 dilution is needed to make a proper scattering solution
PTI Felix GX Horiba version 4.1.0.4096 spectral acquisition program for PTI Time Master Instrument
PTI Time Master Instrument Horiba NA
Pymol Molecular Graphics Program Schrodinger version 2.4
Water bath Thermo Scientific NESLAB RTE 10
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Keywords Extracted From The Given Text:FRET MappingLigand Binding SitesSubunit OrientationConformational ChangesDynamic MotionsBiomolecular SystemLabeling SitesR0 ValuesDonor DyeAcceptor DyeSpectrofluorometerEmission ScanExcitation Wavelength

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Citazione di questo articolo
Northrop, J., Oliver, D. B., Mukerji, I. Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features. J. Vis. Exp. (181), e63433, doi:10.3791/63433 (2022).
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