VJ和HS承认NIH R01 GM094246支持VJ。 HS承认克莱姆森大学创意咨询计划和克莱姆森大学光学材料科学和工程技术中心的创业资金。该项目还得到了墨西哥湾沿岸联盟跨学科生物科学培训凯克中心（NIGMS Grant No.1 T32GM089657-05）的训练奖学金和Schissler基金会DD普通人类疾病转化研究奖学金的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方观点。

所有作者都宣称与本文的内容没有竞争的经济利益。

在这项工作中，提出了使用PIE-MFD单分子FRET实验以高精度对准，校准和测量干涉距离的方案。通过仔细校准所有仪器参数，可以提高测量距离的精度并达到Angstrom精度。为了做到这一点，使用各种多维直方图来分析和识别人群以进一步表征。使用平均宏观时间来验证测量样品的稳定性，可以基于化学计量参数来校正供体和受体光漂白并选择FRET群体。然而，受体的光物理性质可以根据标签的位置而?...

结构生物学研究的一个基本目标是揭示生物分子机器的结构与功能之间的关系。生物分子（ 例如，蛋白质和核酸）的第一个视觉印象发生在20世纪50年代，通过开发X射线晶体学1,2 。 X射线晶体学提供了由晶体包装约束的高分辨率静态结构信息。因此，X射线结构模型的固有不动性可以避免生物分子的动态性质，这是影响大多数生物学功能的因素3,4,5 。核磁共振（NMR） 6,7,8提供了解决水溶液中结构模型的问题的替代方案。一个很大的优势的NMR是其恢复生物分子和构象集合的内在动力学特性的能力，有助于阐明结构，动力学和功能之间的内在关系3,4,5。然而，由样品量和大量样品限制的NMR对较大系统需要复杂的标记策略。因此，迫切需要开发结构生物学中的替代方法。 历史上，Förster共振能量转移（FRET） 9在结构生物学中没有发挥重要作用，因为FRET提供了低精度距离测量的误解。该协议的目的是重新审视FRET确定纳米尺度的距离，使得这些距离可用于构建生物分子的结构模型。第一个实验验证通过测量各种长度的聚合物作为“光谱尺”，1967年10号由Stryer在FRET效率方面的依赖性。在2005年的单分子水平上完成了类似的实验11 。聚脯氨酸分子被证明是非理想的，因此后来使用双链DNA分子12 。这打开了精确距离测量的窗口和使用FRET识别生物分子的结构特性的想法。 当干涉距离范围为〜0.6-1.3 R 0时，FRET最佳，其中R 0是Förster距离。对于在单分子FRET实验中使用的典型荧光团， R 0为〜50。通常，与其他方法相比，FRET在解决和区分结构和动态的能力方面提供了许多优势异质系统：（i）由于荧光的最终灵敏度，单分子FRET实验13,14,15,16可以通过直接计数和同时表征其各个成员的结构来解析异质集合。 （ii）复合反应途径可以在单分子FRET研究中直接解密，因为不需要整体的同步。 （iii）FRET可以及时访问跨越十多年的各种时间领域，涵盖各种各样的生物相关动态。 （iv）FRET实验可以在体外和体内的任何溶液条件下进行 。 FRET与荧光显微镜的组合允许直接在活细胞中研究分子结构和相互作用15,16 ， </<html> sup&gt; 17，18，19 ，甚至高精度20 。 （v）FRET可以应用于几乎任何大小的系统（ 例如，聚脯氨酸低聚物21,22,23,24，Hsp90 25 ，HIV逆转录酶26和核糖体27 ）。 （vi）最后，包含生物分子的所有维度的距离网络可用于导出静态或动态分子的结构模型18,28,29,30,31,32,33,34 ，参考“&gt; 35,36,37。 因此，单分子FRET光谱可用于导出足够精确的距离以用于距离限制结构建模26 。这可以通过利用八维尺寸的荧光信息（ 即激发光谱，荧光光谱，各向异性，荧光寿命，荧光量子产率等）的多参数荧光检测（MFD）28,38,39,40,41,42的优点，宏观时间，荧光强度和荧光团之间的距离）精确而准确地提供距离限制。另外，脉冲交错激励（PIE）与MFD组合（PIE-MFD） 42以监测直接激发受体荧光并选择由含有1：1供体 - 受体化学计量的样品产生的单分子事件。典型的PIE-MFD设置使用连接到共聚焦显微镜主体的双脉冲交错激光激光器，其中光子检测在不同光谱窗口和偏振特性中分为四个不同的通道。更多细节见图1 。 重要的是要注意，FRET必须结合计算方法来实现与FRET结果26,30相符合的类原子结构模型。目前的协议的目的不在于使用FRET衍生距离来构建结构模型的相关方法。然而，这些方法已经与其他技术（ 例如，小角度X射线散射）结合使用或电子顺磁共振），产生了整合结构生物学领域43,44,45,46。目前的目标是为FRET作为结构生物学的定量工具铺平道路。作为示例，该方法用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域（LBD）中的三种构象状态。最终的目标是克服上述限制，并通过提供高精度的测量距离将FRET引入用于结构测定生物分子的综合方法。

<table border="0" cellpadding="0" cellspacing="0" width="672">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">charcoal</td>
			<td style="width:183px;">Merck KGaA</td>
			<td style="width:119px;">K42964486 320</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">syringe filter</td>
			<td style="width:183px;">Fisherbrand</td>
			<td style="width:119px;">09-719C</td>
			<td>size: 0.20um</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">chambered coverglass</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">155409</td>
			<td>1.5 borosilicate glass, 8 wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">microscope cover glass</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">063014-9</td>
			<td>size: 24X60-1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Nuclease free water</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">148859</td>
			<td>nuclease free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">tween-20</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">28320</td>
			<td>10% solution of Polysorbate 20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (High FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">178124895</td>
			<td>5&#180;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (Low FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177956424</td>
			<td>5&#180;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">donor DNA strand</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177951437</td>
			<td>5&#180; -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">DNA strand (No FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td>5&#180; -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">thermal cycler</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:119px;">E6331000025</td>
			<td>nexus gradient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 488 C5&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A10254</td>
			<td>termed cyan-green fluorophore in the manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 647 C2&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A20347</td>
			<td>termed far-red fluorophore in the manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Rhodamine 110</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83695-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Rhodamine 101</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83694-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">LB Broth, Miller</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1426</td>
			<td>For culture of E. coli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Ampicillin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">A0166</td>
			<td>Used at 100 ug/ml final concentration in selective LB medium to maintain plasmid selection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Tetracyline&#160;</td>
			<td style="width:183px;">Calbiochem</td>
			<td style="width:119px;">58346</td>
			<td>Used at 12.5 ug/ml final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Kanamycin</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP906-5</td>
			<td>Used at 15 ug/ml final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Origami B(DE3) Competent Cells</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:119px;">70837-3</td>
			<td>Competent E. coli cells for expression of protein with disulfide bridges</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Isopropyl-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1755</td>
			<td>For induction of E. coli protein expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">HiTrap Chelating HP</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td style="width:119px;">17-0409-01</td>
			<td>For Large-scale FPLC Purification of His-tagged protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Imidazole</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Ni-NTA Agarose&#160;</td>
			<td style="width:183px;">Qiagen</td>
			<td align="right" style="width:119px;">30210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PD-10 Desalting Column</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td style="width:119px;">17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">AktaPurifier</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td align="right" style="width:119px;">28406264</td>
			<td>FPLC Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Dialysis tubing</td>
			<td style="width:183px;">Spectrum labs</td>
			<td style="width:119px;">132562</td>
			<td>15 kD MWCO 24 mm Flath width, 10 meters/roll</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Dichroics</td>
			<td style="width:183px;">Semrock</td>
			<td style="width:119px;">FF500/646-Di01-25x36</td>
			<td>500/646 BrightLight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">50/50 Beam splitter polarizer</td>
			<td style="width:183px;">Qioptiq Linos&#160;</td>
			<td style="width:119px;">G33 5743 000</td>
			<td>10x10 film polarizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Green pass filer</td>
			<td style="width:183px;">Chroma</td>
			<td style="width:119px;">ET525/50m</td>
			<td style="width:167px;">ET525/50m 25 mm diameter mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Red pass filter</td>
			<td style="width:183px;">Chroma</td>
			<td>ET720/150m</td>
			<td>ET720/150m 25 mm diameter mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Power Meter</td>
			<td style="width:183px;">ThorLabd</td>
			<td style="width:119px;">PM200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">UV-Vis spectrophotometer</td>
			<td style="width:183px;">Varian</td>
			<td style="width:119px;">Cary300Bio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorolog 3 fluorometer</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">FL3-22-R3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorohub TCSPC controller</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">Fluorohub-B</td>
			<td>TCSPC electronics for ensemble measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 485L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">485L</td>
			<td>Blue diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 635L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">635L</td>
			<td>Red diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Olympus IX73 Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">IX73P2F</td>
			<td>Microscope frame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 40 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932200, PMA 40</td>
			<td>Optimized for green detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 50 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932201, PMA 50</td>
			<td>Optimized for ed shifter sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">485nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">640nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-640</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Hydraharp 400 and TTTR acqusition software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">930021</td>
			<td>Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">SEPIA II SLM 828 and SEPIA software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">910028</td>
			<td>Laser driver for picosecond pulses , includes SEPIA software controller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">computer</td>
			<td style="width:183px;">Dell</td>
			<td style="width:119px;">optiplex 7010</td>
			<td>cpu: i7-3770 ram:16GB</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">FRET Positioning and Screening (FPS) software</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">MFD suite</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html</td>
		</tr>
	</tbody>
</table>

high precision fret at single-molecule level for biomolecule structure determination

这里介绍了如何在多参数荧光检测（MFD）模式下以单分子水平进行Förster共振能量转移（FRET）进行高精度间距距离测量的协议。 MFD最大化荧光的所有“尺寸”的使用，以减少光物理和实验伪像，并允许在刚性生物分子中以高达±1的精度测量间距距离。该方法用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域的三个构象状态，以解释配体结合时受体的活化。当将已知的结晶结构与实验测量结果进行比较时，对于更多的动态生物分子，他们认为不到3Å。收集覆盖生物分子的整体维度的一套距离约束将使得有可能提供动态生物分子的结构模型ES。

在使用MFD设置（激光线：485nm在60μW和640nm，23μW，5.1节）的典型smFRET实验中，将荧光样品稀释至低皮摩尔浓度（10 -12M = 1pM）并放置在共焦显微镜中，亚纳秒激光脉冲激发标记分子通过激发体自由扩散。典型的共焦体积&lt;4毫升（fL）。在这样低的浓度下，一次只能检测到一个分子。通过目标收集来自标记分子的发射荧光，并使用针孔进行空间过滤。此步骤定义了有效的共聚...

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

本文介绍了单分子水平高精度FRET实验方案。此外，该方法可用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域中的三种构象状态。确定精确的距离是建立基于FRET实验的结构模型的第一步。

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 ...

The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

high-precision-fret-at-single-molecule-level-for-biomolecule

Research

JoVE Journal

Biochemistry

10.6K 次观看.  Clemson University. The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

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

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.

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. ...

科研

生物化学

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

肝糖原分子的提取用于糖原结构测定

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

使用单分子FRET可视化膜受体的构象动力学

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

用单层石墨烯包覆 CryoEM 网格以改善冷冻样品制备

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

作者聚焦：在显微镜网格上使用石墨烯单层增强 CryoEM 样品制备 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

单层石墨烯在冷冻电子显微镜网格中的应用，用于高分辨率结构测定

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...

Methods Article

单分子水平的高精度FRET用于生物分子结构测定