Name: submillisecond conformational changes in proteins resolved by photothermal beam deflection
Uploaded: 2014-02-18T13:00:00
Description: Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

这项工作是由美国国家科学基金会（MCB 1021831，JM）和J.＆E.生物医学研究发展计划（健康的佛罗里达部门，JM）的支持。

1。样品准备<ol><li>进行样品制备和在一个黑暗的房间里所有取样操作，以防止不必要的解笼锁。 </li><li>溶解DM-nitrophen（（1 - （2 -硝基-4,5 -二甲氧基苯基） - N，N，N&#39;，N&#39; -四[（氧羰基）甲基] -1,2 -乙二胺），在50mM HEPES缓冲液，100mM的氯化钾，pH 7.0至400μM的终浓度（ε350nm处 = 4330 M -1 -1 9）。 </li><li>添加氯化钙的0.1M的储液来实现的[Ca 2 +]的所希望的比例?...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoacoustic+calorimetric+study+of+horse+myoglobin.">A photoacoustic calorimetric study of horse myoglobin.</a> Bioph. Chem. 37 (1-3), 73-79 (1990).</li><li>Belogortseva, N., Rubio, M., Terrell, W., Miksovska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+heme+propionate+groups+to+the+conformational+dynamics+associated+with+CO+photodissociation+from+horse+heart+myoglobin.">The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin.</a> J. Inorg. Biochem. 101 (7), 977-986 (2007).</li><li>Mik&#353;ovsk&#225;, J., Suquet, C., Satterlee, J. D., Larsen, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Conformational+Changes+Coupled+to+Ligand+Photodissociation+from+the+Heme+Binding+Domain+of+FixL.">Characterization of Conformational Changes Coupled to Ligand Photodissociation from the Heme Binding Domain of FixL.</a> Biochemistry. 44 (30), 10028-10036 (2005).</li><li>Miksovska, J., Gennis, R. B., Larsen, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothermal+studies+of+CO+photodissociation+from+mixed+valence+Escherichia+coli+cytochrome+bo3.">Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3.</a> FEBS Lett. 579 (14), 3014-3018 (2005).</li><li>Losi, A., Michler, I., G&#228;rtner, W., Braslavsky, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+photolysis+of+caged+calcium:+rates+of+calcium+release+by+nitrophenyl-EGTA+and+DM-nitrophen.">Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen.</a> Biophys. J. 70, 1006-1016 (1996).</li><li>Miksovska, J., Larsen, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+in+metalloproteins.">Structure-function relationships in metalloproteins.</a> Methods Enzymol. 360, 302-329 (2003).</li><li>Miksovska, J., Norstrom, J., Larsen, R. 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背后的光热方法的物理原理是光激发态分子通过振动弛豫消耗多余的能量回到基态，从而导致周围1,12溶剂热供暖。为溶剂，例如水，这将产生一个快速的体积膨胀度（ΔVTH）。激发态分子也可能发生，由于键断裂/形成和/或改变分子结构，可以改变分子（S）（ 即改变范德分子的大小导致非热容积的变化（ΔVnonth）光化学过程范德华体积），以及改变分子（次）从而?...

光热方法，如光声量热法，光热光束偏转（PDB）和瞬态光栅加上纳秒激光激发代表一个强大的替代瞬态光谱学为短命的中间体1,2时间分辨的研究。相反，光学技术，如瞬态吸收和IR光谱，该监测在周围发色团的吸收变化的时间曲线;光热检测方法的热/体积变化的时间相关性，因此是有价值的工具，用于研究的光时访问“沉默”的进程。到目前为止，光声量热法和瞬态光栅已经成功地用于研究光诱导过程的构象动力学包括球蛋白3,4，用氧气传感器蛋白配体相互作用的双原子的配体迁移FIXL 5，电子和血红素-铜质子转运氧化酶6一ND光系统II和光异构化的视紫红质7和8隐构象动力学。 扩大的光热技术所缺乏的内部生色团和/或荧光团的生物系统的应用，PBD技术被结合使用笼形化合物的光致触发几微秒内增加了配位体/底物浓度或更快，这取决于上笼化合物。这种方法允许监控与配体/底物结合到缺乏内部荧光团或生色蛋白，并且在时间尺度不属于商业停流仪器可访问相关的结构性变化动力学和热力学的。这里的PBD的应用监控笼状化合物，钙的热力学2 + DM-nitrophen，光裂解以及动力学的Ca 2 +协会向神经元钙传感器的C端结构域向下流调节元件拮抗剂调制器（DREAM）提出。的Ca 2 +的是光释放的Ca 2 +内10微秒DM-nitrophen和重新绑定到一个unphotolysed笼的〜300微秒的时间常数。另一方面，在apoDREAM的存在下在毫秒级的时间尺度发生的额外动能观察和反映了配体结合的蛋白质。 PBD的应用，探讨在生物系统中的构象转变已经以某种方式限制由于器乐困难;探头和泵浦光束，实现了强大的和可重复的PBD信号，例如艰巨对齐。然而，一个精心设计的仪器设定，温度的精确控制，并在探针和泵浦光束的仔细对准提供一个一致的和健壮的PBD信号，使监控的时间分辨体积和热焓的变化在广泛时间尺度从10微秒至约200毫秒。此外，modific的实验步骤，以保证相同的温度，缓冲液组成，光学元件取向，激光功率等下的样品和参考轨迹的检测的ations显著降低测得的反应体积和焓的实验误差。

<table border="0" cellpadding="0" cellspacing="0" width="639">
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		<tr height="84">
			<td height="84" style="height:84px;width:216px;">1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N&#39;,N&#39;-Tetraacetic Acid</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">D-6814</td>
			<td style="width:220px;">DM-nitrophen, cage calcium compound, keep stock solutions in dark to prevent photodissociation,</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td style="width:84px;">Sigma Adrich</td>
			<td>0909C</td>
			<td>HEPES buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium ferricyanide(III)</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">702587</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:84px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:220px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium chromate</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">307831</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">He-Ne Laser Diode 5mW 635nm </td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">54-179</td>
			<td style="width:220px;">use as a probe beam for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Oscilloscope, </td>
			<td style="width:84px;">LeCroy</td>
			<td style="width:119px;">Wave Surfer 42Xs</td>
			<td style="width:220px;">400 MHz bandwith</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nd:YAG laser</td>
			<td style="width:84px;">Continuum</td>
			<td style="width:119px;">ML II</td>
			<td>pump beam for PBD measurments</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M355; Nd:YAG laser mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">47-324</td>
			<td>laser mirror for 355 nm laser line</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M1 and M2; Laser diode mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">43-532</td>
			<td>visilbe laser flat mirror, wavelength range 300-700 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P1 and P2; Iris Diaphragm</td>
			<td style="width:84px;">Edmind Optics</td>
			<td style="width:119px;">62-649</td>
			<td style="width:220px;">pin hole to shape the probe and pum beams</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">L1; bi-convex lens</td>
			<td style="width:84px;">Thorlabs</td>
			<td style="width:119px;">LB1844</td>
			<td style="width:220px;">a lens to focus the probe beam at the detector, EFL 50 mm, wavelength range 350 - 2000 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DM, dichroic mirror</td>
			<td style="width:84px;">Thorlab </td>
			<td style="width:119px;">DMLP505</td>
			<td style="width:220px;">a longpass dichroic mirror with a cutoff wavelength of 505 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F1; Edge filter</td>
			<td style="width:84px;">Andower </td>
			<td style="width:119px;">500FH90-25</td>
			<td>a long pass filter with a cutoff wavelength of 500 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-controlled cuvette holder</td>
			<td style="width:84px;">Quantum Northwest</td>
			<td style="width:119px;">FLASH 300</td>
			<td></td>
		</tr>
	</tbody>
</table>

submillisecond conformational changes in proteins resolved by photothermal beam deflection

光热光束偏转与光声量热法和热光栅一起属于监视光诱导微秒的蛋白质构象变化用传统的站到毫秒时间尺度是不能访问的时间分布体积和焓变光热方法的家庭流量仪表。另外，由于在体积和/或焓的整体变化进行了探讨，这些技术可以应用到那些缺乏荧光团和或发色团标记的蛋白质和其他生物大分子。为了监测与钙有关的结构变化动力学和热力学+结合钙的传感器，例如神经元钙传感器，笼中的钙化合物，DM-nitrophen，是用来光电触发快速（τ&lt;20微秒）增加游离钙浓度和相关的量和焓变是使用光热光束偏转技术探讨。

PBD的一个代表性的例子痕迹的Ca 2 +的光释放的Ca 2 + DM-nitrophen示于图3。快速相位对应于光裂解的Ca 2 + DM-nitrophen与Ca 2 +释放的，而慢相体现的Ca 2 +结合到nonphotolysed笼。样品PBD振幅缩放到参考化合物的振幅随着温度依赖系数[C Pρ/（的dn / dt）的]的函数的快和慢相的积因子，根据公式4和缩放所观察到的量释放到溶液中，非热容...

Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

在这里，我们报告的光热光束偏转技术在同一个笼中钙化合物，DM-nitrophen，监控微秒和毫秒动态及与钙的关联关联到一个神经元的钙传感器，下游调控元件拮抗剂调制器结构变化热力学相结合的应用。

亚毫秒构象变化蛋白的光热光束偏转已解决

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

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