我们感谢她的资料的使用，北京吴。这项工作是支持由美国国家科学基金会职业奖JEK

1。 〜50 nm的直径小（越野车）Unilamellar囊泡膜蛋白质折叠的制备<ol><li> 1,2 - dimyristoyl - SN -甘油- 3 - phosphocholine（DMPC）在氯仿中血脂的解决方案是购买和aliquotted在20毫克每小瓶数量存储到干净的玻璃小瓶。添加到每一瓶氮气层，以防止脂质过氧化，与瓶盖和封口膜密封小瓶。小瓶存放在-20℃冷冻，直到使用。 </li><li>一个包含20毫克脂质在氯仿分装的单瓶是用于每个实验。小瓶的内容使用1小时，直到无溶剂残留的氮气流干。 </li><li>干血脂悬浮于1毫升20 mM磷酸钾缓冲液（pH值= 7.3）使用水浴sonicator为〜30秒。这种暂停脂质的解决方案会出现阴天，是一个圆锥形的底部转移至15毫升塑料管。添加到另一个磷酸盐缓冲液1毫升等分，原本载脂的空玻璃瓶，和超声是重复的解决方案，然后添加到相同的15毫升管。重复此过程，共4次，直到最后管量为4毫升，导致血脂浓度在5毫克/毫升在这股囊泡解决方案。 </li><li>脂质囊泡溶液4毫升的量是摆在一个温暖的（〜30 ° C）水浴和超声占空比为50％，使用1小时ultrasonicator微尖。水浴的目的是双重的。首先，它可以防止由于超声过程变得太热脂质的解决方案。其次，温水浴，确保水脂解温度仍然在超声双层相转变温度以上; DMPC，这一转变温度为〜23 ° C。 </li><li>超声解决方案是通过孔径为0.22μm注射器过滤器删除从sonicator尖端的碎片，这个过滤解决方案是平衡的，在37℃过夜° C。 </li></ol> 2。初始荧光展开曲线的样品制备<ol><li>是由一个20毫米的磷酸盐缓冲液（pH值7.3）10 M尿素原液。重要的是，水被添加到固体（反之亦然），因为大量的尿素在解决方案中占有显着的体积。此尿素溶液，可溶质加入到一个干净的，空瓶子，并加水，达到了预期的最终体积。这个解决方案可能需要水浴中搅拌溶解的溶质的一个热点板块升温。尿素是吸湿的，因此，实际尿素原液的浓度应确定使用折射率。 </li><li>一个股票缓冲液20 mM磷酸盐（pH值7.3）。 </li><li> A股解决方案展开蛋白准备。 8 M尿素，20毫米的磷酸盐缓冲液中，这股蛋白质的解决方案应该有一个〜200微米蛋白的浓度。 </li><li>在下列方式中的荧光研究的样品准备。股票蛋白的相应卷（2.3节），股票脂解（5毫克/毫升的脂质，第1条），股票10米的尿素溶液（2.1节），和股票磷酸盐缓冲液（2.2节）相结合，使样品中含有〜4 μM蛋白质，1毫克/毫升的血脂，0到1米递增8 M尿素。每个样品的总体积为200μL。包括一个例子试算表（表1）列出卷需要使用200微米的股票蛋白质溶液。 </li><li>空白样品如在2.4节中所述，然而，蛋白质是不添加。在蛋白质的地方，可能会增加18米的尿素溶液4μL，使其他组件的浓度在2.4节相同的。这些空白减去散射和荧光光谱在蛋白质中出现的其他背景信号。 </li><li>从第2.4和2.5的样品可以在37 ° C孵育荧光光谱测量前至少2个小时，以确保完成折叠和平衡。 </li></ol> 3。荧光光谱的测量每个样品和空白的色氨酸荧光测量采用稳态荧光。应记录激发波长290纳米的荧光光谱，以避免激发酪氨酸残基，并从305到500纳米扫描。典型的入口和出口带通为3纳米。波长增量和积分时间，可以优化信号信噪比。波长增量和集成时间的典型值是1海里/步骤和0.5秒/步，分别的。膜蛋白折叠成合成脂类，只有当温度高于双层相变温度。因此，在这种情况下DMPC与，样品的温度恒定在30 ° C。在这些前利用一个微量熔融石英160μL的能力比色皿periments。 4。新一代的初步展开曲线和吉布斯自由能展开蛋白膜的估算<ol><li>荧光光谱在一个XY数据集的形式装入伊戈尔临，MATLAB，原产地或Excel等软件。 </li><li>必须减去从尿素和脂质囊泡的背景信号，使用下列程序： 频谱=蛋白原料的荧光光谱中存在的脂质囊泡和特定浓度的尿素。 频谱乙=原料相应的空白样品的荧光光谱包含脂质囊泡和相同的尿素浓度，在频谱;没有在空白样品的蛋白质。 更正频谱=频谱A - C *频谱B. C是一个标量通常等于1.0。在某些情况下，C是小于或大于1.0，因为散射造成过度的问题，或减〜305-320 nm区域附近。 </li><li>最大荧光的波长，λ最大，载列从纠正每个尿素浓度的光谱。一个典型的价值为充分展开，而折叠的蛋白质〜330 nm的膜蛋白是350纳米。表格的波长转换为分数展开转换λ 最大值 （〜330〜350纳米）范围内的范围从0.0到1.0关联λ最大值展开人口的一小部分。例如，一个λ330 nm处的最大价值对应为0.0，350 nm的对应到1.0，和340 nm处的对应分数为0.5展开。这种折叠蛋白的一小部分，然后对尿素浓度作图。如上所述，尿素浓度应通过测量折射率的实验来确定。 </li><li>我们假设，该蛋白可以存在于两种状态之一：折叠或展开。分数情节的展开，F，与尿素浓度，C，可拟合方程： 2 <img src="/files/ftp_upload/2669/2669eq2.jpg" alt="公式（2）" /> 系数 m对应的自由能的变化率与变性剂浓度，C M对应的中点折叠人口等于展开人口的尿素浓度。这些值是由最小二乘法拟合得到的数据分析软件的装修变量（0.001987千卡/摩尔•K），R是气体常数，T是温度（303 K）。 </li><li>从拟合得到的系数是用来确定尿素的情况下展开的Gibbs自由能（kcal / mol的）： <img src="/files/ftp_upload/2669/2669eq3.jpg" alt="公式3"/> 。 </li></ol> 5。吉布斯自由能展开更多的精密测量曲线优化展开。 <ol><li>上文所述的展开曲线揭示了尿素浓度的范围，导致在λ最大的变化最大。这个范围通常很小，这个小范围内开展的大部分发生。为了准确地确定开展的免费能源，额外的样品进行了分析，这一地区获得一个阴谋，它包含许多数据点优化最小二乘法拟合的必要。例如，如果蛋白质在2和4 M尿素之间展开，样本可包含2 - 4米增量为0.2 M尿素，以更准确地揭示这一重要地区的展开曲线的形状。这是没有必要在每个尿素浓度来衡量的空白光谱，它是足以衡量一个空白频谱每〜0.5 M步。 </li><li>从原始的展开曲线获得从这个情节的展开自由能获得可能略有不同，但反映了一个更精确的的值。 </li></ol> 6。代表性的成果： <table border="1"><tbody><tr><td>样品</td><td>决赛[尿素]，男</td><td>股票蛋白（微升） </td><td>股票脂质（微升） </td><td>股票10 M尿素（微升） </td><td>股票缓冲区（微升） </td></tr><tr><td> 1 </td><td> 〜0.16 </td><td> 4 </td><td> 40 </td><td> 0 </td><td> 156 </td></tr><tr><td> 2 </td><td> 1 </td><td> 4 </td><td> 40 </td><td> 20 </td><td> 136 </td></tr><tr><td> 3 </td><td> 2 </td><td> 4 </td><td> 40 </td><td> 40 </td><td> 116 </td></tr><tr><td> 4 </td><td> 3 </td><td> 4 </td><td> 40 </td><td> 60 </td><td> 96 </td></tr><tr><td> 5 </td><td> 4 </td><td> 4 </td><td> 40 </td><td> 80 </td><td> 76 </td></tr><tr><td> 6 </td><td> 5 </td><td> 4 </td><td> 40 </td><td> 100 </td><td> 56 </td></tr><tr><td> 7 </td><td> 6 </td><td> 4 </td><td> 40 </td><td> 120 </td><td> 36 </td></tr><tr><td> 8 </td><td> 7 </td><td> 4 </td><td> 40 </td><td> 140 </td><td> 16 </td></tr></tbody></table>股票的解决方案，使荧光样品量见表1 。 <img src="/files/ftp_upload/2669/2669fig1.jpg" alt="图1" /> 图1〜5微米的具有代表性的膜蛋白，包含一个单一的色氨酸残基的色氨酸荧光光谱。 （一）原蛋白的荧光光谱（频谱从4.2）;（二）原料空白的荧光光谱（从4.2频谱二），（三）更正4.2频谱。 <img src="/files/ftp_upload/2669/2669fig2.jpg" alt="图2" /> 更正大量的尿素浓度从图1图2。色氨酸膜蛋白的荧光光谱。 <img src="/files/ftp_upload/2669/2669fig3.jpg" alt="图3" /> 图3。展开适合在4.4式，从图2中的数据获得的曲线。吉布斯自由能计算根据4.5节。

<ol>
	<li>Shirley, B. A., Shirley, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Folding and Stability. , 177-190 (1995).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+and+Analysis+of+Urea+and+Guanidine+Hydrochloride+Denaturation+Curves.">Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves.</a> Methods Enzymol. 131, 266-279 (1986).</li><li>Lau, F. W., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Assessing+the+Stability+of+a+Membrane+Protein.">A Method for Assessing the Stability of a Membrane Protein.</a> Biochemistry. 36, 5884-5892 (1997).</li><li>Burgess, N. K., Dao, T. P., Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Barrel+Proteins+that+Reside+in+the+Escherichia+coli+Outer+Membrane+in+Vivo+Demonstrate+Varied+Folding+Behavior+in+Vitro.">&#946;-Barrel Proteins that Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro.</a> J. Biol. Chem. 283, 26748-26758 (2008).</li><li>Booth, P. J., Curnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+scene+investigation:+membrane+proteins.">Folding scene investigation: membrane proteins.</a> Curr. Opin. Struct. Biol. 19, 8-13 (2009).</li><li>Hong, H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+coupling+of+integral+membrane+protein+stability+to+lipid+bilayer+forces.">Elastic coupling of integral membrane protein stability to lipid bilayer forces.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4065-4070 (2004).</li><li>Sanchez, K. M., Gable, J. E., Schlamadinger, D. E., Kim, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Tryptophan+Microenvironment,+Soluble+Domain,+and+Vesicle+Size+on+the+Thermodynamics+of+Membrane+Protein+Folding:+Lessons+from+the+Transmembrane+Protein+OmpA.">Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA.</a> Biochemistry. 47, 12844-12852 (2008).</li></ol>

目前的协议描述了一代展开膜相关的蛋白质和多肽含有色氨酸残基的曲线。在这里，它是假设，色氨酸荧光反映蛋白质折叠和插入到合成脂质囊泡，或在溶液中展开。额外的假设，如两个国家的折叠和自由能与变性剂浓度的线性关系，在当前的报告;。修改这些假设的结果， 在不同的方程式2实验协议可以很容易地进行修改，以利用不同的光谱观测，如圆二色性，吸收，或从外在染料的荧?...

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thermodynamics of membrane protein folding measured by fluorescence spectroscopy

膜蛋白质折叠是一个新课题，根本和健康相关的意义。膜蛋白在细胞的基础需要全面研究，无处不在的家庭这种蛋白质折叠的丰度。此外，在我们的能力来表征与错误折叠的蛋白质相关疾病的进步有上进心重大的理论和实验的努力，在蛋白质折叠领域。不幸的是，阻碍在这一重要领域的快速进步与膜蛋白折叠机制的复杂性相关的内在挑战。在这里，我们列出了测量变性，ΔG ° H 2 O的情况下发生的吉布斯自由能的热力学性质的实验过程，一个代表从E组成的膜蛋白大肠杆菌 。该协议的重点，作为变性剂浓度的功能的折叠和展开状态，以确定均衡的人口应用荧光光谱。合成脂质囊泡以及在数据分析过程中的关键步骤准备实验考虑突出。这种技术是多才多艺的，可与不同类型的变性剂，包括温度和pH值，以及在各种折叠血脂和胶束环境进行。目前的协议，是一个可以推广到任何膜或可溶性蛋白，符合标准的设置，下面讨论。

Watch this Scientific Journal Video about Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy at JoVE.com

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

此视频文章详细介绍了实验过程中色氨酸荧光膜蛋白质折叠吉布斯自由能获得。

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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SCIENTISTS IN ACTION

18.2K 次观看.  University of California San Diego - UCSD. 此视频文章详细介绍了实验过程中色氨酸荧光膜蛋白质折叠吉布斯自由能获得。

视频: Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

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生物工程

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

细胞外基质蛋白纤维的布里渊谱的制备

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

分子弹簧常量分析由生物元原力探针光谱

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

荧光蛋白中脯氨酸类似物对脯氨酸的残基特异换：骨架的“分子手术”如何影响折叠和稳定性

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Methods Article

荧光光谱法测定膜蛋白质折叠的热力学