Name: collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms
Uploaded: 2011-04-07T11:00:00
Description: Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

这项工作是由加拿大卫生研究院研究院（CIHR），国家科学与工程研究理事会（NSERC）和CIHR培训资助奖学金（LF）的支持。我们感谢教授杰拉德D.赖特（麦克马斯特大学，加拿大）（6）为AAC - II表达质粒。

1。准备库存解决方案<ol><li>净化大分子的兴趣。 （在这种情况下，氨基糖苷类的N - 6&#39; -乙酰基- II（AAC6&#39;二），是孤立的报道13。） </li><li>准备4公升的透析液。 （在这种情况下，我们使用25毫米4 - （2 - 羟乙基）- 1 - piperazineethanesulfonic酸（HEPES，MW​​ 238.3克/摩尔），含有2毫米乙二胺四乙酸（EDTA，292.2兆瓦），在pH值7.5。） </li><li>透析的AAC（6&#39;）- II的样品在400微米（5毫升），用?...

<ol>
	<li>Cliff, M. J., Ladbury, 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=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, 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=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, 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=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, 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=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. 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Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

这个变量- C管件的分析部分以前已经10详细描述。在这里，我们报告收集适合这种方法的变量- C的数据集的实际问题。重要的是，所有的蛋白质与配体样本是从同一只股票方案的制定。因此重要的是，准备充足的库存解决方案，初步完成了整个系列实验。这将确保比例的AAC（6&#39;）- II和AcCoA是不变的，所有的实验中，并降低浓度样本，以抽样方式随机波动。 在这种情?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
</tr>		</tbody>
		</table>
		</div>

collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

等温滴定量热（ITC）是常用的确定与主机高分子配体的结合相关的热力学参数。 ITC主机/配体的相互作用研究一些较常见的光谱方法的优点。例如，两个组件交互时释放或吸收的热量是直接测量，不需要任何外源性的记者。因此，结合焓和协会常数（Ka）直接获得从国际贸易中心的数据，可用于计算熵的贡献。此外，等温线的形状是依赖于C值和所涉及的机理模型。 C -值被定义为C = N [P] TKA，[P] T是蛋白浓度，n是主机内配体结合位点的数目。在许多情况下，为一个给定的配体的多个结合位点非等效ITC允许每一个人的结合位点的热力学约束力参数的表征。然而，这需要使用正确的绑定模型。这种选择可能会产生问题，如果不同型号，可容纳相同的实验数据。我们曾表明，这个问题是可以规避在几个C -值进行实验。同时适合不同的模型在不同的C -值获得的多个等温线。正确的模式是未来整个变量- C数据集的拟合善良的基础上确定。这个过程是应用氨基糖甙类抗生素导致耐药酶氨基糖苷类的N - 6&#39; -乙酰基转移酶- II（AAC（6&#39;）- II）。虽然我们的方法是适用于任何系统，更好地证明这一战略的必要性与高分子配体变构或协同系统，和不同的结合模式提供相同的数据基本上是相同的适合。据我们所知，有没有市售的这类系统。 AAC（6&#39;）- II，是一个同源二聚体，含有两个活性部位，呈现出两个亚基之间的协同。然而，在一个单一的C -价值得到国际贸易中心的数据，可以适合同样至少有两个不同型号的两集网站独立模型和两站点顺序（合作）模式。通过不同的C值，如上面所述，它是建立正确的绑定模型的AAC（6&#39;）- II是两个站点的顺序的绑定模型。在这里，我们描述的，必须在执行国际贸易中心实验时所采取的的，为了获得适合变量- C分析数据集的步骤。

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

ITC是一个功能强大的工具，为研究其主机的一个配体的结合。然而，在复杂的系统的几种模式可能适合同样的数据。这里介绍的方法，提供了一种手段来澄清合适的绑定模型和复杂的系统，提取相应的热力学参数。

收集可变浓度等温滴定量热数据集，以确定约束机制

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

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