Name: thermodynamics of membrane protein folding measured by fluorescence spectroscopy
Uploaded: 2011-04-28T15:00:00
Description: Watch this Scientific Journal Video about Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy at JoVE.com

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...

<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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<td>DMPC</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>850345C</td>
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<td>Urea</td>
<td>&nbsp;</td>
<td>MP Biochemicals</td>
<td>04821527</td>
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<td>Potassium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P288</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
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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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