Name: structural information from single-molecule fret experiments using the fast nano-positioning system
Uploaded: 2017-02-09T12:30:00.000+00:00
Duration: 12 min 30 s
Description: Watch this Scientific Journal Video about Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System at JoVE.com

The authors thank B. Gruchmann for the mechanical drawings of the flow chamber. Further, we want to express our gratitude to Max Beckers and Florian Drechsler for insightful comments and discussions regarding NPS and the underlying sampling engine.

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The authors have nothing to disclose.

我们目前的设置和实验程序准确地确定通过挠性接头到生物大分子， 例如 ，核酸和/或蛋白质附着染料之间的FRET效率。 为了确保精确的smFRET测量（第3节），关键的是在测量过程中的任何时间，以排除从流动室的空气。此外，确保不超载荧光流室。荧光团必须明确区分开来，以确保正确的分析。作为smFRET对，其不显示供体的漂白必须从分析中排除，确保在视场中的分子的&gt; 80％的在电影结束漂白。考虑到样品的β因子和γ-因子中的不均匀性，纠正供体和受体通道的串扰和相对的检测效率，分别计算每个FRET对个别。 有关荧光光谱仪（第7至9）的测量的信号强度与已记录的数据的光谱分辨率之间的良好折衷有被发现。为此在荧光光谱仪的激发和发射通路狭缝必须适应依赖于所使用的仪器和样品的浓度。 此外，我们目前的快速NPS分析方法来获得短暂的或动态MACROM的结构信息olecular复合物。 NPS已应用于以显示非模板DNA链的路径和转录起始因子在古细菌RNA聚合酶开放的复杂的位置。使用超过60种不同的距离测量网络，我们发现，快速NPS，配备了新实施的采样引擎（Eilert，T.，贝克尔，M.，德雷克斯勒，F.，与米氏，J.筹建中），减少了由幅度≈2订单所需此复杂smFRET网络的分析，由于相对于原来的全球NPS方法27的时间。该算法的鲁棒性是植根于都市中之吉布斯采样与并行回火方案相结合。快速NPS显示确切的网络结果的可重复性并与早期30日公布的结果一致。 几种不同的方法已经发表，旨在从smFRET测量11推断的结构信息</s向上&gt;，12，13，14，15，16，17，18。所有这些方法只提供一个特定染料模型。由此，染料，即不符合由各个模型所做的假设，不能使用，或者导致错误的结构信息。快速NPS中，与此相反，可以选择每个染料分子的不同的模型。这有助于解释两者的不同构象的行为时，染料分子本身，以及用于它的连接的连接体。染料分子的局部分子的环境中，以及它的物理性质将决定哪种模式是最合适的。 为古细菌起始复合物的分析smFRET网络，对于所有染料分子的各向同性假定导致急剧下降我n作为相比于经典模型的可信卷的大小。在具有动态位置平均为所有染料组合分子所有可信体积尺寸的中位数（95％）降低到小于0.5nm 3。然而，这些染料分子后验不再符合其smFRET测量，表明假设而导致虚假的结构信息。与此相反，在经典模型所确定的后验是与确定smFRET效率一致。 由于各向同性和/或动态位置求得所有染料的假设导致不一致，快速NPS使其中每个染料可分配的五款车型之一染料分子前科。每个型号都使用相同的访问卷。用于染料的AV计算的算法做了几个假设。首先，荧光团的空间形状由球形近似。因此，一个直径考虑到荧光团的WID次，高度和厚度应当使用（第12节）。此外，连接体的形状是由一柔性杆近似。第12节给出的数值计算了染料Alexa的647通过12-C接头附着。迄今为止，这是不可能精确地确定先验哪种模式是最适合给定的实验的几何形状，并且因此所有的模型进行测试。在一般情况下，人会选择这使可能的最小后尺寸的模型，同时仍然与数据一致。要测试的车型可以选择是否与smFRET数据是一致的，我们都计算后和可能性。一致性意味着90％以上的从后回收的样品中是可能性的95％置信区间内。 虽然这是事实，该染料分子的各向异性低，较小的距离的不确定性，在一个smFRET网络几何安排也必须加以考虑。因而人，而representing染料分子与一个iso模型低荧光各向异性是典型的首选，一致性检验提供了选择正确的染料模型更直接的手段。染料模型的最佳选择可以导致定位精度急剧增加，并在同一时间保持与它的FRET数据网络的一致性。 概括地说，快速NPS允许获得大的大分子复合物的结构和动态信息。在对比共同的结构的方法，如X射线晶体学或低温电子显微镜这允许监测高度灵活的或瞬态络合物，从而大大扩大了复杂的生物过程的机理的理解。

确定的生物分子的结构对于理解其功能的重要前提。两个用于结构测定行之有效的方法是低温电子显微镜和X-射线晶体学1,2。如今，这两种方法提供一个分辨率下到埃级高分辨率的结构信息。这两种方法已被广泛用来阐明大生物分子的结构，例如蛋白质复合物。虽然现有的方法都不断被整个过去的几十年改善，生物结构的复杂性，仍对结构生物学的一大挑战，尤其是当大，动态和短暂络合物进行了研究3。 为了研究大分子复合物的动力学，特别是结构与功能的关系，单分子的方法有省被提供的有用信息4。一些新的战略被开发提供有关收购的结构和动态信息的正交方式。实例是高速AFM 5，机械操作6，荧光定位显微镜7，以及单分子荧光共振能量转移（smFRET）8,9。由于相当早就FRET已称为分子尺 ，由于对生物大分子10的长度尺度的距离依赖性。 smFRET的一个特别有趣的应用是使用从smFRET测量获得的距离信息来推断结构信息11，12，13，14，15 </sup&gt;，16，17，18，19，20，21，22，23。由于smFRET的高时间分辨率，蛋白质结构的移动部分的位置可以本地化。然而，为了从约染料分子smFRET数据重要校正参数提取量化信息所需要的测量24中被确定。与这些校正因子，FRET的效率E FRET可以使用公式来计算<img alt="式（1）" src="/files/ftp_upload/54782/54782eq1.jpg" /> ， 在那里我 A和I D </sUB&gt;分别是供体和受体分子，的荧光强度（参见图2）。的β因子占串扰，供体发射的漏入受体信道和由下式计算<img alt="公式（2）" src="/files/ftp_upload/54782/54782eq2.jpg" />在那里我是A和I&#39;D是供体的荧光强度和受体分子的光漂白后的受体分子。 的γ因子校正两个通道中的相对探测效率的差异，以及在该供体的荧光量子产率和受体染料的差异。它是由每个人的时间跟踪的计算方法请注意，本说明书中忽略了受体分子，其有时变得很重要，并需要为待校正以及直接激励。用于确定这些修正因子是有用的，以便光的物理变化和结构动力学区分以激励这两个供体以及受体以交替的方案25。 为了不仅获得定量smFRET效率而且定量的结构信息，所述纳米定位系统（NPS）于2008年26被引入。名称是根据其相似性基于卫星的全球定位系统（GPS）的选择。该NPS是结合smFRET和X射线晶体学数据在生物大分子复合物不明染料位置的定位的混合技术。的Crystal结构用作基准帧和smFRET结果用于得到未知的荧光团的位置（ 天线 ），并从晶体结构（ 卫星 ）已知的位置之间的距离信息。在连续实验中，天线和多个卫星之间的距离被测量和天线的位置是由基于贝叶斯参数估计统计学上严格的分析方案的方法来确定。其结果，不仅天线的最可能的位置被计算，但其完整的3D不确定性分布，即所谓的后，由可信卷可视化。此外，NPS扩大到允许完整smFRET网络27的分析。 所述NPS已被用来解决一些在真核转录的重要问题，上游的DNA，非模板DNA和RNA聚合酶II延伸共内新生mRNA的即过程mplex 12，28，也显示出转录起始的影响因素26和一个开放式启动子的动态建筑群29。此外，NPS是用来阐明古细菌的RNA聚合酶开放的复杂30转录起始因子TFE的位置，这竞争结合相同位点的转录延伸因子Spt4 / 5 31的，特别是结构。 从那时起，许多基于smFRET结构方法已经发表15，18，21，23。当比较不同的基于smFRET结构的方法，它变得清晰，该方法的明显的精度在很大程度上取决于染料模型的特定选择。应该注意的是染料分子可能根据当地环境表现出不同的空间和取向行为。 为此，快NPS引入32。快速NPS采用了先进的采样算法大大减少计算时间。此外，快速NPS允许一个执行结构分析，并为每个染料分子，用户可以从一组五个不同染料的模型将在下面描述选择。最保守的模式，堪称经典之作，假设染料只占用一个，但不明，地位。在该位置，所述荧光团可以一个锥体，其尺寸从其相应（时间依赖性）荧光各向异性测定内自由转动。圆锥体的取向是未知的，转换测量smFRET效率成距离时导致大的不确定性。在这方面，该模型是保守的，因为这将导致最小精度相比，其他染料模式LS。只有很短的距离应该由经典机型率先作出一个明显不正确的位置确定的假设。对于典型smFRET价值观，正确的立场是始终封闭在较大音量可信。 然而，由于较高的精度是可取的，它开发和测试的替代染料模型，这可能有助于改善精度是重要的。如果染料旋转比其固有的荧光寿命快得多，所谓异模型可以应用。这里，取向因子κ2（所需要的计算特性各向同性福斯特半径<img alt="式（1）" src="/files/ftp_upload/54782/54782eq4.jpg" /> ）设定为2/3。其结果是，相比那些在经典机型的32可信计算量几乎是两个数量级较小。在于不仅能够快速reori该荧光团是在环境中发现的情况下entation，但另外的快速运动都在其访问的卷，应使用meanpos异模型。在此模型中，将染料有效地只占用一个平均位置，其中所述空间平均是由一个多项式距离转换15占。如果例如（通常疏水的）染料附着到亲水性区域， 例如，该DNA该模型适用。所述meanpos异模型的应用导致通过大约两个因素中的可信卷的尺寸进一步减小。然而，联系到蛋白质的染料可能会可逆地结合在其空间上访问的卷（AV）数的疏水补丁。这些区域之间的瞬时开关，但在一个区域发生自由转动和快速的局部运动，最好由无功meanpos异模型中描述的荧光团。对于类似的情况，其中染料是不能自由旋转VAR-meanpos模式适用。多个D-<html>有关这些车型etails可以在我们最近发表的32被发现。 这些模型提供了专门占了染料可能遇到的各种环境和应用它们明智地优化其定位精度丰富的剧目。在快速NPS附加到特定位置每染料分子可以被分配到一个单独的模式，使得FRET的伙伴被允许有不同的型号。这使得无限的贴近自然造型。然而，重要的是，一个执行严格的统计测试，以确保由最终模型组合获得的结果仍然与实验数据一致。这些测试包括在快速NPS软件。 为了快速NPS适用于实验数据是必需的（只）三个输入参数的测量。首先，该染料对特定各向同性福斯特半径（/54782/54782eq5.jpg"/&gt;）已被确定，因此，供体染料的量子产率（QY），供体荧光发射光谱和受体的吸收光谱需要测量，这些测量可以在进行散装，使用标准的光谱仪和一个标准的荧光光谱。对于每一对，则R 0然后使用免费软件PhotochemCAD计算，并且可以在NPS分析中使用。此外，（时间分辨）染料分子的荧光各向异性需要使用一个极化（和时间）敏感荧光光谱仪来获得。然而，对于快速NPS的最重要的输入参数是在单分子荧光显微镜的设置测量的smFRET效率，如一个全内反射荧光显微镜（TIRFM） 。 在这里，我们提出了一个一步一步的协议获得smFRET数据和应用快速NPS（ 图1）。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Flowchamber preparation&lt;/strong&gt;</td></tr><tr><td>Customized metall sample holder</td><td>self-built</td><td>n/a</td><td></td></tr><tr><td>quartz-glass slides, 76&amp;nbsp;mm x 26&amp;nbsp;mm</td><td>Technical Glass Products</td><td>26007</td><td></td></tr><tr><td>coverslips, 60 mm x 24&amp;nbsp;mm</td><td>Marienfeld</td><td>101242</td><td></td></tr><tr><td>detergent, Hellmanex II</td><td>Hellma</td><td>320.001</td><td></td></tr><tr><td>ultra-pure water from Synergy UV</td><td>Millipore</td><td>2512600</td><td></td></tr><tr><td>Zepto plasma cleaner</td><td>Diener</td><td>n/a</td><td></td></tr><tr><td>(3-aminopropyl)-triethoxysilane, p.a.</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>methoxy PEG-succinimidyl valerate, 5 kDa</td><td>Laysan Bio Inc.</td><td>MPEG-SVA-5000-1g</td><td></td></tr><tr><td>biotinylated PEG-succinimidyl valerate, 5 kDa</td><td>Laysan Bio Inc.</td><td>BIOTIN-PEG-SVA-5000</td><td></td></tr><tr><td>Sodium biocarbonate</td><td>Sigma-Aldrich</td><td>S5761 Sigma&amp;nbsp;</td><td></td></tr><tr><td>Sodium carbonate</td><td>Sigma-Aldrich</td><td>S2127 Sigma-Aldrich&amp;nbsp;</td><td></td></tr><tr><td>sealing film (Nescofilm)</td><td>Fisher Scientific</td><td>12981805</td><td></td></tr><tr><td>Tygon Flexible Silicone Tubing, 0.8&amp;nbsp;mm ID, 2.4&amp;nbsp;mm OD</td><td>Saint-Gobain Performance Plastics</td><td>720958</td><td></td></tr><tr><td>Fine-Bore Polyethylene Tubing, 0.58 mm ID, 0.96 mm OD (Smiths Medical)</td><td>Fisher Scientific</td><td>12665497</td><td></td></tr><tr><td>Neutravidin</td><td>Life Technologies</td><td>A2666</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Total internal reflection fluorescence microscope&lt;/strong&gt;</td></tr><tr><td>Nd:YAG Laser, 532 nm</td><td>Newport Spectra-Physics</td><td>EXLSR-532-100-CDRH</td><td></td></tr><tr><td>diode-pumped solid-state laser, 491 nm, Calypso</td><td>Cobolt</td><td>904010050</td><td></td></tr><tr><td>diode laser 643 nm, iBeam smart</td><td>Toptica</td><td>iBEAM-SMART-640-S</td><td></td></tr><tr><td>dichroic mirror, 532 RDC</td><td>Chroma</td><td>F33-540</td><td></td></tr><tr><td>dichroic mirror, 476 RDC</td><td>Chroma</td><td>F33-476z</td><td></td></tr><tr><td>acousto-optic tunable filter</td><td>AA Opto-Electronic</td><td>AOTFnC-VIS</td><td></td></tr><tr><td>plano-convex cylindrical lens, f = 75 mm</td><td>Thorlabs</td><td>LJ1703L1-A</td><td></td></tr><tr><td>plano-concave cylindrical, f = -300&amp;nbsp;mm</td><td>Thorlabs</td><td></td><td></td></tr><tr><td>prism, PS 991</td><td>Thorlabs</td><td>PS991</td><td></td></tr><tr><td>focusing lens, f = 75&amp;nbsp;mm</td><td>Thorlabs</td><td>LA1608-B</td><td></td></tr><tr><td>syringe pump, PHD 2000</td><td>Harvard Apparatus</td><td>70-2002</td><td></td></tr><tr><td>2 stepper motors, Z812B</td><td>Thorlabs</td><td>Z812B</td><td></td></tr><tr><td>piezoelectric actuator, PE4</td><td>Thorlabs</td><td>PE4</td><td></td></tr><tr><td>IR diode laser</td><td>Edmund Optics</td><td>CPS808</td><td>part of the autofocus system</td></tr><tr><td>dichroic mirror, 775 DCXR</td><td>Chroma</td><td>775 DCXR</td><td></td></tr><tr><td>position-sensing detector (PSD), PDP90A</td><td>Thorlabs</td><td>PDP90A</td><td>part of the autofocus system</td></tr><tr><td>water-immersion objective, Plan Apo 60X WI, NA 1.2</td><td>Nikon</td><td></td><td>MRD07601</td></tr><tr><td>dichroic mirror, 645 DCXR</td><td>Chroma</td><td>645 DCXR</td><td>part of the emission pathway</td></tr><tr><td>emission filter, 3RD550-510</td><td>Omega Optical</td><td>3RD550-510</td><td>green channel in the emission pathway</td></tr><tr><td>emission filter, 3RD660-760</td><td>Omega Optical</td><td>3RD660-760</td><td>red channel in the emission pathway</td></tr><tr><td>EMCCD camera, iXon+ DU897EBV</td><td>Andor</td><td>AND-20-00032</td><td></td></tr><tr><td>EMCCD camera, iXon3 DU897D-BV</td><td>Andor</td><td>AND-20-000141</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Miscellaneous&lt;/strong&gt;</td></tr><tr><td>Varian 50</td><td>Cary</td><td></td><td>UV-VIS spectrometer</td></tr><tr><td>Fluorolog2</td><td>SPEX</td><td></td><td>fluorescence spectrometer</td></tr><tr><td>Solis (V4.15)</td><td>Andor</td><td></td><td>control software for the EM-CCD camera</td></tr><tr><td>Apt user utility&amp;nbsp; (V1.022)</td><td>Thorlabs</td><td></td><td>control software for the piezo-motors</td></tr><tr><td>Norland Optical Adhesive 68</td><td>Thorlabs</td><td></td><td>adhesive</td></tr><tr><td>PC-AFN-0.8 Nile red</td><td>Kisker Biotech</td><td></td><td>avidin-coated fluorescent multispec beads&amp;nbsp;</td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td>technical computing language for custon written software</td></tr><tr><td>Origin (V9.0)</td><td>Originlab</td><td></td><td>scientific graphing and data analysis software</td></tr><tr><td>Hellma 105-202-15-40</td><td>Hellma</td><td>105-202-15-40</td><td>absorption cuvette of 1&amp;nbsp;cm path length</td></tr><tr><td>Hellma 105-251-15-40</td><td>Hellma</td><td>105-251-15-40</td><td>fluorescence cuvette with 3 mm path length</td></tr></tbody></table>

structural information from single-molecule fret experiments using the fast nano-positioning system

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

转录基因表达的第一步中的所有生物。在古菌，转录由单个RNA聚合酶（RNAP）进行。相比于真核生物，古细菌RNAP有着惊人的相似结构，同时具有更简单转录机器的真核同行。因此，古细菌可以用作模型系统通过RNA聚合酶II（RNA聚合酶II）来研究真核转录起始。近日，古细菌RNA聚合酶开放的复杂的完整的体系结构已经从单分子荧光共振能量转移和NPS确定。从NPS分析的数据被用来构建完整的古开放的启动子复合物，它提供了有益的见解转录起始机制的典范。 为了阐明这种结构，分别位于开放的启动子COM中未知的天线染料分子之间测量smFRET效率复杂和已在该RNAP，其位置由晶体结构（PDB-ID:2WAQ）是已知的五个参考网站包含了一些已知的卫星染料分子37。天线染料被装上的非模板DNA，TFB，TBP或TFE的不同位置中的任一个。在这项研究中所用的完整网络包括超过60测量的距离。 图7描绘了从NPS分析建完整的古细菌开放启动子复合物的模型。它包括双链启动子的DNA（亮和暗蓝色），该RNA聚合酶（灰色）和转录起始因子TBP（紫色），TFB（绿色）和TFE（黄色）。该模型叠加从NPS分析，可信册，其中采用了经典模型（A），ISO模型（B）计算的，meanpos-ISO模式（C）的VAR-meanpos异模型结果（D）和VAR-meanpos<html>模型（E）。 <img alt="图1" src="/files/ftp_upload/54782/54782fig1.jpg" /> 图1: 所需快速NPS计算参数的采集和处理的工作流程。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54782/54782fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54782/54782fig2.jpg" /> 图 2: 事件smFRET的示范性荧光强度实时跟踪。 smFRET，II:供体（绿色）和示出了三个特征阶段，即我的受体分子（红色）的荧光强度施主荧光受体漂白后，三:施主漂白后的背景荧光。g2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54782/54782fig3.jpg" /> 图3: 流动室供smFRET实验的示意图。流室安装到与亚克力玻璃持有人定制的金属支架。流室的夹层设计包括一个石英玻璃（熔融石英）有两个孔用于安装入口和出口管道，密封膜和关闭所述流动室盖玻片滑动。为TIRF照明棱镜被安装到所述流室的下半部。空心螺丝卡提供流室入口和出口。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54782/54782fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img<html> ALT ="图4"SRC ="/文件/ ftp_upload / 54782 / 54782fig4.jpg"/&gt; 图4: 在石英玻璃载玻片和密封膜的制备。石英玻璃载玻片的机械制图表示的孔的位置（以毫米为单位给出）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54782/54782fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54782/54782fig5.jpg" /> 图5: 流动室机械制图。对于铝棱镜支架的措施，有机玻璃支架和铝安装帧毫米给出。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54782/54782fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> igure 6"SRC ="/文件/ ftp_upload / 54782 / 54782fig6.jpg"/&gt; 图6: 用于smFRET实验棱柱型的TIRF设置的示意图。缩写光学元件:A，孔; DM，分色镜;楼发射滤波器; L，镜头;男，镜; 0，客观的; P，棱镜; PSD中，位置敏感光电二极管; S，样品; PS，定位阶段; T，望远镜。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54782/54782fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54782/54782fig7.jpg" /> 图7: 不同的模型假设的仿真结果。所有的图片显示的古细菌RNA聚合酶（PDB ID:2WAQ，顶视图）与模型为启动子DNA（分别TDNA和ntDNA在蓝色和青色，）一起，TBP（紫色），TFB（绿色）和TFE（黄色）在古开复30。可信卷叠加的（A）的经典模型，（B）ISO模型，（ 三 ）meanpos异型号，（ 四 ）VAR-meanpos异模型和（E）变种的NPS模拟结果-meanpos模式。所有卷都在68％可信度所示。经典和VAR-meanpos网络与smFRET数据一致。相反，在所有染料选择了ISO，meanpos异或VAR-meanpos异模型网络与实测数据不一致。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54782/54782fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System at JoVE.com

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

从单分子结构信息FRET实验使用快速纳米定位系统

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

structural-information-from-single-molecule-fret-experiments-using

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

12.1K 次观看.  Ulm University. We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

视频: Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

工程学

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

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 &#945;-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 &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-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.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

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

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

生物工程

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...