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：[DM-nitrophen]。与K d的蛋白质的Ca 2 +协会大于10μm，内[Ca 2 +]的比值：[DM-nitrophen]为1:1，优选为防止光释放的Ca 2 +到出笼DM-nitrophen绑定。的确，考虑到对于DM-nitrophen对K d值为为10 nM和DM-nitrophen与Ca 2 +的总浓度为400 mM的，〜90％的钙结合蛋白具有K D = 10μM将在apoform。另一方面，对于Ca 2 +的研究为K D &lt;10μM的蛋白质结合，这是优选的，以减少内[Ca 2 +]：[DM-nitrophen]比为0.95，以防止Ca 2 +的络合与脱辅基事先笼光离解蛋白质。 </li><li>溶解参考化合物，K 3的[Fe（III）（CN）6]或Na 2的CrO 4，在相同的缓冲液作为样品。 </li></ol> 2。设置实验<ol><li>基本的实验配置示于图2。 </li><li>使用一个销孔（P 2）的探测光束的（He-Ne激光器的632 nm的输出，〜5毫瓦的激光功率）的直径调节到1毫米，并通过单元的中心放置在温度传播的探测光束用M 1镜控制单元支架。 </li><li>用一个反射镜（M 2）的样本后面居中探测光束对位置敏感检测器的中心。 </li><li>着眼于这样的方式，顶部的两个二极管和底部的两个二极管以及两个二极管的检测器的左侧和右侧之间的电压差之间的电压差检测器的中心的探测光束为零。 </li><li>随后，塑造一个直径泵浦光束，Q开关Nd的355纳米输出：采用3毫米的针孔（P 1）放置在两个355 nm的激光反射镜之间的YAG激光器，半高宽5纳秒）。 </li><li>通过比色皿的中心Copropagate泵浦光束表现在图2。重要的是，这两个激光束通过光学单元中几乎共线的方式的中心传播到获得可测量的偏转角，从而高PBD信号的振幅。在实验条件下，探针和泵光束的交叉点的角度小于15°。 </li><li>使用参考化合物对齐探针和泵束达到令人满意的PBD信号， 即在更长的时间尺度（〜100毫秒）的良好S / N比和稳定的PBD振幅。 </li><li>通过355纳米的激光反射镜的增量调节调节泵的光束的位置相对于所述探测光束。 </li><li>测量PBD参考信号的幅度作为位置敏感探测器上的两个顶部和底部的光电二极管之间的一个差异。该PBD信号应该具有在快速时间量程（&lt;10微秒）的振幅的快速增加，并保持稳定的100毫秒时间尺度表现在图3。拍摄到的PDB振幅的拍摄变异性是内信号幅度的5％，且信号的再现性主要受振动。 </li><li>检查在PBD信号振幅的线性相对于所述释放热能，通过测量PBD信号对激励激光功率和关于爱因斯坦的数量线性相关吸收，E 一=（1-10-A），其中A对应的激发波长参考吸光度。 </li><li>遵守以下的激光功率约为1,000μJ，并在激发波长小于0.5，以防止多光子吸收和减少泵束的功率，分别为样品/参照化合物的吸收，并确保PBD信号的线性度。 </li></ol> 3。 PBD测量<ol><li>开始的PBD痕迹的参考测量值。将参照化合物的溶液在1.0厘米×1.0厘米或1.0厘米×0.5厘米石英池，并在该温度控制架的细胞定位。这两种路径长度提供类似的PBD幅度。 </li><li>从16-35℃的温度增量为3°C检测的基准PBD信号作为温度的温度范围内的一个函数</li><li>在每次温度变化，检查位置灵敏探测器的探测光束的位置，并重新如果需要调整位置到检测器的中心。 </li><li>根据等式2检查PBD信号的线性度作为的函数[（的dn / dt）的/ C Pρ]术语。 </li><li>将样品溶液相同的光学元件作为在用于保持光学元件的同一方向作为基准测量的参比化合物。 </li><li>检测样品的PBD的痕迹在相同的温度范围作为参考，并检查样品的PBD振幅的线性相对于[（DN / DT）/ C Pρ]术语。 </li></ol> 4。数据分析偏转的幅度成正比的量的变化，由于根据式（1）的样品加热（ΔVth）和非热容量变化（ΔVnonth）： <img src="/files/ftp_upload/50969/50969eq1.jpg" alt="式（1）" /> 样品（A S的幅度）和参考（A R）PBD信号可以用方程2和3，分别进行说明。 <img src="/files/ftp_upload/50969/50969eq2.jpg" alt="公式2" /> <img src="/files/ftp_upload/50969/50969eq3.jpg" alt="式（3）" /> PBD信号是正比于仪器的响应参数（K）和爱因斯坦的数量被吸收（E a）所示 。在等式2中的第一项，（的dn / dt）的（1/ρCP）Q，对应于信号的变化，由于释放到溶剂中的热量。将dn / dt项表示温度依赖性变化的折射率，ρ是该溶剂的密度，C p是比热容。所有参数是已知的蒸馏水和缓冲溶液通过在蒸馏水中并在合适的缓冲液的参照化合物比较一PBD信号可以被确定。 Q为热报酬e的量d，将该溶剂。在ρ（DN / Dρ）项是一个无单位的常数，它是与温度无关的温度范围从10-40℃下10。的Δn 绝对术语对应于折射率的变化，由于吸光物质的溶液中的存在和它的可忽略不计，如果探测光束的波长位移相对于任何种类的溶液的吸收光谱。从参考化合物（A R）而产生的信号由公式3，其中E Hν是光子能量的激发波长，表示E Hν= 80.5千卡/摩尔为355 nm激发。 <ol><li>就拿参考PBD信号的幅度表现在图3的预触发和后触发的PBD信号之间的差值。以类似的方式，决定了快（ 一个 S 快 ）的幅度和慢相（A 公司 慢样品PBD信号）。 </li><li>消除仪器响应参数，K，由PBD信号为基准的幅度缩放样本PBD信号的振幅。样品信号与参考信号的比值给出方程4和可写为： <img src="/files/ftp_upload/50969/50969eq4.jpg" alt="公式4" /> </li><li>利用这个公式，确定释放到溶液（Q）和非热容量变化（ΔVnonth）自的[一个曲线的斜率和截距，分别有一个光引发反应相关的（A S / A R）E中的热ħν]长期与温度相关项[（DN / DT）（1/ρCp）]。 </li><li>为了确定反应体积和焓变的快速和缓慢的过程，根据公式5-7缩放观察量和焓变到相应的量子产率。 <img sRC =“/ files/ftp_upload/50969/50969eq5.jpg”ALT =“公式5”/&gt; <img src="/files/ftp_upload/50969/50969eq6.jpg" alt="公式6" /> <img src="/files/ftp_upload/50969/50969eq7.jpg" alt="公式7" /> 对于一个多步过程与动力学发生在10微秒〜200毫秒之间的时间尺度，体积和与该反应的各个步骤相关的焓的变化可以被确定。的振幅和寿命的各个步骤由数据拟合的函数F（t）描述该卷和焓变化的时间曲线进行分析。 <img alt="公式8" src="/files/ftp_upload/50969/50969eq8.jpg" /> 其中，α0对应于A 的快速和αi对应至一个S 慢为每个单独的过程和τi分别是单个反应步骤的寿命。从速率常数FO的温度依赖性Ř单个进程（K I = 1 /τi）该活化焓和熵的参数可以容易地使用的Eyring曲线来确定。 </li></ol>

<ol>
	<li>Gensch, T., Viappiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+photothermal+methods:+accessing+time-resolved+thermodynamics+of+photoinduced+processes+in+chemistry+and+biology.">Time-resolved photothermal methods: accessing time-resolved thermodynamics of photoinduced processes in chemistry and biology.</a> Photochem. Photobiol. Sci. 2, 699-721 (2003).</li><li>Larsen, R. W., Mik&#353;ovsk&#225;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+resolved+thermodynamics+of+ligand+binding+to+heme+proteins.">Time resolved thermodynamics of ligand binding to heme proteins.</a> Coord. Chem. Rev. 251 (9-10), 1101-1127 (2007).</li><li>Westrick, J. A., Peters, K. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+Thermodynamic+Changes+Photoinduced+in+5,12-trans-locked+Bacteriorhodopsin.+Evidence+that+Retinal+Isomerization+is+Required+for+Protein+Activation.">Time-resolved Thermodynamic Changes Photoinduced in 5,12-trans-locked Bacteriorhodopsin. Evidence that Retinal Isomerization is Required for Protein Activation.</a> Photochem. Photobiol. 72, 590-597 (2000).</li><li>Kondoh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Induced+Conformational+Changes+in+Full-Length+Arabidopsis+thaliana+Cryptochrome.">Light-Induced Conformational Changes in Full-Length Arabidopsis thaliana Cryptochrome.</a> J. Mol. Biol. 413 (1), 128-137 (2011).</li><li>Kaplan, J. H., Ellis-Davies, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolabile+chelators+for+the+rapid+photorelease+of+divalent+cations.">Photolabile chelators for the rapid photorelease of divalent cations.</a> Proc. Natl. Acad. Sci. U.S.A. 85 (17), 6571-6575 (1988).</li><li>Eisenberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equation+for+the+Refractive+Index+of+Water.">Equation for the Refractive Index of Water.</a> J. Chem. Phys. 43 (11), 3887-3892 (1965).</li><li>Ellis-Davies, G. C., Kaplan, J. H., Barsotti, R. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+profiles+for+CO+photodissociation+from+heme+model+compounds:+effect+of+proximal+ligands.">Thermodynamic profiles for CO photodissociation from heme model compounds: effect of proximal ligands.</a> Inorg. Chem. 44 (4), 1006-1014 (2005).</li><li>Dhulipala, G., Rubio, M., Michael, K., 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=Thermodynamic+profile+for+urea+photo-release+from+a+N-(2-nitrobenzyl)+caged+urea+compound.">Thermodynamic profile for urea photo-release from a N-(2-nitrobenzyl) caged urea compound.</a> Photochem. Photobiol. Sci. 8, 1157-1163 (2009).</li></ol>

作者什么都没有透露。

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">
	<colgroup>
		<col />
		<col />
		<col /> 
		<col />
	</colgroup>
	<tbody>
		<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 | Chemistry | Video

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 ...

submillisecond-conformational-changes-proteins-resolved-photothermal

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8.9K 次观看.  Florida International University. 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。 

视频: Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

科研

化学

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

使用气相色谱怀孕期间分析脂肪酸组成的变化在大鼠肝组织

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

利用 iccd 摄像机对无氧环境下的三重收集有机化合物进行光物理表征

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&lt;sup&gt;&amp;#8722;&lt;/sup&gt;

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

改进 x 射线激光时分辨序列飞秒晶体学微晶的高粘度挤出

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...