PLD担任加拿大蛋白质工程研究主席。这项工作由加拿大卫生研究所向民盟提供的赠款资助。这项工作还得到了日本科学促进协会（JSPS）和日本生物导向技术研究促进机构（BRAIN）的科学研究资助。我们感谢克里斯·马歇尔博士和迈克·奎珀博士开创性的工作，导致FIPA。我们还感谢萨凯·齐达博士为这项工作提供设施，并感谢劳里·格雷厄姆博士为荧光灯激发和排放过滤器的建立提供便利。

1. 生长单一冰晶
<ol><li>拿一个干净的金属锅（直径15厘米，高4.5厘米），适合，可以漂浮在乙烯乙二醇冷却浴。</li>
	<li>准备聚氯乙烯 （PVC） 圆柱形模具，直径 4.5 厘米，高 3-4 厘米，厚 4 毫米），从管道锯断面。<ol>
<li>一侧切一个档次（1毫米宽，2毫米高）（图1A）。</li>
		<li>准备尽可能多的模具，可以舒适地放入锅（图1B）。 注:一项研究表明，聚乙烯醇（PVA）可以影响冰核22。然而，在我们的开放模具PVC系统中，我们没有遇到非典型冰层的问题。</li>
	</ol>
</li>
	<li>将真空润滑脂的光膜涂在每个模具的底部表面环上，即切出缺口的表面。将这个涂有油脂的凹面密封在金属平底锅上，凹槽定位于远离平底锅中心的地方。小心不要用油脂填充或阻塞凹槽。</li>
	<li>将 0.22 μm 过滤和脱气/除离子水添加到锅的中心，但在模具之外，并允许水通过凹槽慢慢进入模具。小心不要引入任何泡沫。水层应约5毫米深。</li>
	<li>将平底锅放入温度控制的乙二醇浴池中，冷却至-0.5 °C。 平底锅应该是完全水平的。如有必要，在平底锅两侧添加压载重量。</li>
	<li>锅和水达到 -0.5 °C 后，在锅中间和模具外加入一小块冰。<ol>
<li>这将核化冰在超冷水中的生长，穿过锅和模具。每个模具底部的小缺口只能通过一个冰晶传播，导致每个模具中只有一个冰晶。</li>
		<li>隔夜孵化形成一层冰。</li>
	</ol>
</li>
	<li>在接下来的三天里，每天向每个模具添加 13 毫升 4 °C 的脱气/除离子水，并在每增加一天后将乙二醇浴池的温度降至 -0.8 °C，第二天降至 -1.1 °C，第三天降至 -1.5 °C。<ol>
<li>在那些温度下孵化过夜。</li>
		<li>到第四天，模具应该完全装满冰。</li>
	</ol>
</li>
	<li>将模具从平底锅中拉下来，将冰晶从模具中推出，并存放在干净的表面（如称重船）中，在 -20 °C 冰柜中存放约 1 小时，然后再处理。</li>
	<li>与其准备许多小的单一冰晶，一个体积几升的大单冰晶可以在一个恒温孵化器中准备，正如骑士23所描述的。如果将大冰晶保存在 -15 至 -20 °C 的冰柜中，则可储存一年或更长期。单晶部分可根据需要用锯从冰块上切开。</li>
</ol>2. 确定冰晶的奇点和方向
<ol><li>通过在冰柜室中观察两个交叉的极性（图1D）来确定从模具中排出的冰是否是单个晶体。<ol>
<li>如果冰晶是单一的，则不应看到任何裂缝或不连续性，冰晶内的光向不应发生变化。 注意:如果没有冰柜室，可以在所有需要的步骤中使用冷室，谨慎地快速工作并谨慎处理冰。</li>
	</ol>
</li>
	<li>由于冰的裂口，可以同时确定 c轴的方向。使用以下信息确定方向:<ol>
<li>当 c轴与事件光完全平行时，理论上，没有光会穿过交叉的极性。如果事件光的标题与 c轴平行，则当光在交叉极性之间旋转时，均匀的多色光谱通过晶体传输。这种均匀的发射产生于冰Ih沿其 c轴24的光学不活动。冰晶的基底平面对 c轴是正常的。</li>
		<li>当 c轴与事件光进一步平行，冰晶在交叉极性之间旋转时，传输的光将每旋转 90°，传输的光将交替 0-100% 传输。</li>
		<li>最常见的情况是 ，c轴对圆柱形冰晶的圆形平面是正常的。</li>
	</ol>
</li>
	<li>通过将冰晶紧紧包裹在铝箔中，用针头将一个小孔戳入基底平面上的冰（对c轴正常），并将其置于真空中 20 分钟，从而确定 a 轴的方向。<ol>
<li>这种治疗将在基底平面上产生六边形对称性的蚀刻，其中A轴穿过六面星的顶点（图2A）。</li>
		<li>如果需要，冰晶可以用锯子平行或垂直于六边形的一侧切割，分别用垂直于冷手指的主棱镜或次要棱镜平面安装它（图3）。</li>
	</ol>
</li>
</ol>3. 将荧光标签防冻蛋白吸附到单一冰晶中
<ol><li>将单个冰晶安装到冷手指 上（图1C），首先将一个腔钻进水晶顶部。为此，用两根直径稍异但与冷指直径相似的铝棒交替融化冰层，形成冷手指可以容纳的腔。</li>
	<li>将冷手指冷却至-0.5 °C，放在冰腔中，并将冰晶保持原位，直到它冻结到金属（图2B）。将晶体连接到手指时避免气泡，因为它们会阻碍热量从手指到棒的有效传输。</li>
	<li>用过滤过的除离子水或缓冲器填充一个直径约为冰晶两倍的半球杯，冷却至约 4 °C。 将冰块冰晶浸入杯中，去除多余的水或缓冲液，使冰晶顶部与液层大致水平，冰不会接触杯壁。<ol>
<li>用绝缘材料盖住杯子，将温度降低到-5°C。</li>
		<li>等待大约1小时，冰晶形成一个半球，检查其状态大约每20分钟（图2C）。</li>
		<li>冰会通过融化和生长冰晶来形成半球杯的形状，但触摸杯壁绝不应过度生长。墙壁和半球之间至少应该有1厘米的缝隙，冷手指不应该从冰上伸出来。</li>
	</ol>
</li>
	<li>从冰晶中取出杯子，将荧光蛋白溶液加入最终体积为 25-30 毫升，并达到所需的分析浓度，小心保持杯中液体总体积不变。典型的 AFP 浓度为 0.1 毫克/毫升。<ol>
<li>将冰晶重新注入杯中，使冰晶顶部与液体处于水平，冰晶不会接触杯壁（图2D）。</li>
		<li>将冷手指温度降至 -8 °C，让蛋白质溶液冻结到冰晶中 2-3 小时，经常搅拌溶液。蛋白质溶液形成的冰在阻止冰生长之前应至少为5毫米。</li>
	</ol>
</li>
	<li>从杯子中取出冰晶，同时仍然附着在冷手指上。通过将冷却剂通过冷手指加热到略高于 0 °C，将冰从后者分离，并等待冰晶融化。</li>
	<li>将水晶平坦的一面放在干净的表面上，如称重盘，小心不要触摸新形成的冰，在交接前至少存放在-20°C。</li>
</ol>4. 防冻蛋白冰平面的可视化
<ol><li>荧光的可视化是在较暗的冰柜或冷室进行的，通过将冰晶平坦的一面放在带有波长特定激发滤光片的灯下，以激发荧光标签和相机发射过滤器以阻止任何其他非特定光。根据图案，可以估计受AFP约束的冰平面（图4）。</li>
	<li>如果没有波长特定的灯，则可以使用紫外线灯箱。</li>
	<li>传统的冰蚀刻也可以简单地通过允许冰半球在-20°C升华至少3小时，之后残留蛋白粉可能会在冰面上可见（图5）。</li>
	<li>可以重复第 2.3 步，以确定完成的冰半球 的斧头的方向。</li>
</ol>

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F., 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=Structural+basis+for+the+superior+activity+of+the+large+isoform+of+snow+flea+antifreeze+protein.">Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.</a> Biochemistry. 49 (11), 2593-2603 (2010).</li><li>Takamichi, M., Nishimiya, Y., Miura, A., Tsuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+active+qae+isoform+confers+thermal+hysteresis+activity+on+a+defective+sp+isoform+of+type+iii+antifreeze+protein.">Fully active qae isoform confers thermal hysteresis activity on a defective sp isoform of type iii antifreeze protein.</a> FEBS J. 276 (5), 1471-1479 (2009).</li><li>Bar-Dolev, M., Celik, Y., Wettlaufer, J. S., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+ice+growth+and+melting+modifications+by+antifreeze+proteins.">New insights into ice growth and melting modifications by antifreeze proteins.</a> J. R. Soc. Interface. 9 (77), 3249-3259 (2012).</li><li>Celik, Y., 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=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (4), 1309-1314 (2013).</li><li>Garnham, C. P., Campbell, R. L., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dimeric+beta-helical+model+of+an+ice+nucleation+protein+with+bridged+active+sites.">Novel dimeric beta-helical model of an ice nucleation protein with bridged active sites.</a> BMC Struct. Biol. 11, (2011).</li></ol>

未声明任何利益冲突。

查尔斯·奈特研制的冰蚀方法，用于确定法新社的冰平面，大大推进了对AFP冰结合机理的研究。虽然AFP的结构可以通过X射线晶体学26，27 来解决，但没有明显的方法来推断AFP绑定在冰上的互补表面。当I型法新社从冬季比目鱼的最初特征，它被假设绑定到冰28的主要棱镜平面。然而，奈特的破冰实验I型法新社显示，他们与冰13金字塔平面结合。这一结果促使法新社研究界重?...

抗冻蛋白（AFPs）的产生是生活在冰层中环境中的一些生物体的重要生存机制。直到最近，人们还认为AFP的唯一功能是防止或减缓内部冰晶的生长，这些冰晶会阻断循环，造成组织损伤和渗透应激。不能容忍任何程度的冰冻的生物，如鱼，表达AFP，以完全抑制冰晶生长1。其他，如草，是耐冻和表达AFP，以抑制冰的再封装，减少形成大冰晶在他们的组织2。在低温下稳定膜是另一个功能，建议为AFP3。最近，一个新的角色被建议为法新社的南极细菌， 马里诺莫纳斯原始，从冰覆盖的咸水湖4。这个AFP是一个更大的粘合蛋白5的一部分，被认为是附加细菌到冰上，以更好地获得氧气和营养物质6。其他微生物已知分泌AFP，这可能改变它们生活7的冰的结构。
在一些鱼类、昆虫、植物、藻类、细菌、硅藻和真菌中发现了AFP。它们具有显著不同的序列和结构，与不同祖先在不同场合的进化相一致:然而，它们都与冰结合，并通过吸附抑制机制8抑制其生长。AFP各有一个特定的表面，作为其冰结合点（IBS）。这些通常通过9-11表面残留物的现场定向突变来识别。IBS 假设将水分子排列在与特定冰平面相匹配的冰样图案中。因此，法新社形成其配体之前，绑定到它5，12。冰飞机可以通过米勒指数来定义，不同的AFP可以与不同的平面结合。因此，I型法新社从冬季比目鱼绑定到20-21金字塔平面13，III型法新社结合主棱镜和金字塔平面使用复合冰结合表面11，14，而云杉芽虫法新社，一个过度活跃的法新社，同时结合到主平面和基底平面15，16。其他过度活跃的AFP，如 MpAFP，与多个冰平面结合，如它们对单个冰晶半球5，17的完全覆盖所示。据推测，超活性AFP结合基底平面和其他飞机的能力，可能占其活性的10倍，比适度活跃的AFP18。虽然超活性AFP的效率是有据可查的，但它们与多个冰层结合的能力仍然不为人所知。
确定法新社飞往冰上飞机的最初方法是由查尔斯·奈特13，19开发的。在这种方法中，一个宏观的单一冰晶被安装在一个空心金属棒（冷手指）上，通过将其浸入装满脱气水的半球杯中形成半球。然后，半球被淹没在AFP的稀释溶液中，在几个小时内，由通过冷手指循环的乙二醇温度控制，从法新社溶液中长出一层冰到冰晶半球。冰晶从溶液中取出，从冷手指上分离出来，放置在-10至-15°C的冰柜室中。表面用锋利的刀片刮去防冻蛋白溶液的冷冻表面薄膜，冰晶允许升华至少3小时。升华后，由AFP绑定的冰平面可视为从残余蛋白质中提取的白色蚀刻图案。冰半球可以定向到其 c轴和 斧头，定位冰的基础平面和棱镜平面，并确定蚀刻补丁的米勒指数。
在这里，我们描述了对确定法新社绑定冰平面的原始方法的修改，我们称之为荧光基冰平面亲和力（FIPA）11。AFP 的荧光标记要么带有钟音标签，如绿色荧光蛋白 （GFP）11，16，17，20，要么与荧光染料共价绑定到 AFP5，21。荧光标记的AFP被吸收到一个冰晶中，并且使用与原始冰蚀实验相同的实验程序过度生长。在整个实验中，可以使用紫外线（UV）灯监测与生长的冰半球结合的AFP的程度。实验完成后，半球可以直接从冷手指上取下并成像，无需升华。然而，如果需要，半球可以留给升华，以可视化传统的冰蚀刻。对FIPA方法的修改将传统的冰蚀协议缩短了几个小时。此外，还有可能同时成像几个具有不同荧光标签的 AFP，以可视化 AFP 绑定冰平面的重叠模式。

<table><tbody><tr><td>NESLAB RTE Refrigerating Bath/Circulators</td><td>Thermo Scientific</td><td>RTE7</td><td></td></tr><tr><td>Ethylene glycol, Premixed Antifreeze/Coolant</td><td>Certified</td><td>29-3037-0</td><td>Common automotive antifreeze</td></tr><tr><td>Cold finger</td><td>not available</td><td>not available</td><td>Custom made with brass (9 cm long, 1.5 cm outer diameter)</td></tr><tr><td>Hemispherical cup</td><td>not available</td><td>not available</td><td>Custom made with resin (8 cm outer diameter, 6 cm inner diameter)</td></tr><tr><td>High Dual Output Lighting System</td><td>Lightools Research</td><td>LT-99D2, Illumatools DLS 120 volts AC, LT-9470FX, LT-9549FX</td><td>Additional and custom excitation filters can be purchased from Lightools Research</td></tr><tr><td>Camera</td><td>Canon</td><td>EOS 50D</td><td></td></tr><tr><td>Emission Filters</td><td>Lightools Research</td><td>LT-9EFPVG, LT-9GFPVG, LT-9RFPVG</td><td>Filter ring adapter may be required to fit filter onto camera lens</td></tr></tbody></table>

determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity

防冻蛋白 （AFP） 表现在各种冷硬生物体中，以防止或减缓内部冰的生长。AFP 通过冰结合表面与特定的冰平面结合。基于荧光的冰平面亲和力 （FIPA） 分析是一种用于确定 AFP 与之结合的冰平面的修改技术。FIPA 基于确定法新社绑定冰平面的原始冰蚀方法。它在缩短的实验时间内产生更清晰的图像。在 FIPA 分析中，AFP 被荧光标记为有幻想标记或共价染料，然后慢慢融入宏观单一冰晶中，该晶体已预制成半球，并定向以确定 a 轴 和 c 轴。法新社的冰半球在紫外线下被成像，使用过滤器来阻挡非特异性光，以可视化法新社的平面。AFP 的荧光标签允许实时监控 AFP 吸附到冰中。已发现这些标签不会影响 AFP 绑定的平面。FIPA 分析还引入了将多个不同标签的 AFP 绑在同一个单一冰晶上的选项，以帮助区分其绑定平面。FIPA的这些应用有助于增进我们对AFP如何与冰结合以阻止其生长的理解，以及为什么许多产生AFF的生物会表达多种AFP等形。

单个冰晶的准备和安装是 FIPA 程序的两个步骤，其中最常出错。确定准备好的冰晶是否为单一的，通过交叉极性体（图1D）检查它，如协议部分的第2.1步所概述。如果用于 FIPA 分析的多晶冰晶，则结果将是非晶体在半球的不连续绑定，而没有连贯的结合模式 （图 6B）。如果冰晶接口位于法新社绑定的冰层周围，最终结果可能无法解释。因此，在进入下一步之前，应仔细检...

Watch this Scientific Journal Video about Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity at JoVE.com

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

防冻蛋白 （AFP） 与特定的冰平面结合，以防止或减缓冰的生长。基于荧光的冰平面亲和力 （FIPA） 分析是对确定 AFP 绑定冰平面的原始冰蚀刻方法的修改。AFP 采用荧光标记，并融入宏观单一冰晶中，并在紫外线下可视化。

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 ...

determining-ice-binding-planes-antifreeze-proteins-fluorescence-based

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

9.0K 次观看.  Queen's University. 防冻蛋白 （AFP） 与特定的冰平面结合，以防止或减缓冰的生长。基于荧光的冰平面亲和力 （FIPA） 分析是对确定 AFP 绑定冰平面的原始冰蚀刻方法的修改。AFP 采用荧光标记，并融入宏观单一冰晶中，并在紫外线下可视化。

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

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

科研

化学

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

生物化学

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

映射的适体对ATP的结合部位使用MicroScale程序热泳

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.

一种用于研究冰和包合物水合物结晶的微流控方法

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

生物学

Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

Source: Jung, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/58763">High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy.</a> J. Vis. Exp. (2019).
This video describes high-performance fluorescence anisotropy that helps to monitor the interaction between transcription factors and DNA. The assay monitors the interactions of a fluorophore-labeled DNA molecule with its specific transcription factor by measuring the degree of polarization due to molecular rotation or anisotropy of the fluorophore-labeled DNA.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

摘要

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LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

荧光各向异性作为一种工具来研究蛋白质相互作用

映射的适体对ATP的结合部位使用MicroScale程序热泳

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

一种用于研究冰和包合物水合物结晶的微流控方法

蛋白质 - 配体相互作用采用差示扫描荧光法测定

用于确定转录因子-DNA 结合亲和力的荧光各向异性

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

摘要

探索更多视频

此视频中的章节

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

荧光各向异性作为一种工具来研究蛋白质相互作用

映射的适体对ATP的结合部位使用MicroScale程序热泳

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

一种用于研究冰和包合物水合物结晶的微流控方法

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

用于确定转录因子-DNA 结合亲和力的荧光各向异性

蛋白质 - 配体相互作用采用差示扫描荧光法测定