Name: helical organization of blood coagulation factor viii on lipid nanotubes
Uploaded: 2014-06-03T13:45:00
Description: Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

10SDG3500034和UTMB-NCB启动资金，SSM：这项工作是由美国心脏协会国家科学家开发资助。作者承认冷冻电镜和科学计算设施的西利结构生物学中心在UTMB（ <a href="http://www.scsb.utmb.edu">www.scsb.utmb.edu</a> ），以及博士。史蒂夫Ludtke和Ed Egelman的帮助，二维和三维螺旋重建算法。

1，样品制备<ol><li>缓冲液交换的人FVIII-BDD 14和猪FVIII-BDD 15对HBS-钙缓冲液（20mM HEPES，150 mM氯化钠，5mM的氯化钙 ，pH值= 7.4）并浓缩至1.2毫克/毫升。保持蛋白溶液于-80℃。 </li><li>通过为1:4 w / w的比例在氯仿混合半乳糖（GC）和磷脂酰丝氨酸（PS）准备脂质纳米管（LNT）。蒸除氯仿在氩气下并溶解在HBS缓冲液中的脂质为1毫克/毫升。保持LNT的解决方案，在4°C。 </li></ol><p class="...

<ol>
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在此工作的方法呈现高度同源性的蛋白质的2膜结合的组织之间进行区分：人和猪FVIII自组装于在人体中所遇到的条件下的脂质纳米管。 在描述的过程中，人类和猪FVIII成功举办螺旋状脂质纳米管，这是最关键的一步。下一个关键步骤是通过快速冷冻在接近液态氮的温度保持在样品中非晶薄的冰。保留在无定形冰和液氮温度下的样品保持水合的螺旋...

凝血因子VIII（FVIII）是由六个域2,332个氨基酸的糖蛋白大：A1-A2-B-A3-C1-C2 3。经凝血酶激活的FVIII作为内与膜结合的tenase复合物的辅因子以因子IXa。激活的FVIII（FVIIIa）来的FIXa中的膜-视方式结合增强FIXa的蛋白水解效率超过10 5倍，这是有效的凝血4是至关重要的。尽管重要作用的FVIII在凝固和tenase复合物的形成中起着，功能膜结合的FVIII结构尚未得到解决。 为了解决这个问题，单脂双层碳纳米管（LNT）富含磷脂酰丝氨酸（PS），能够结合的FVIII以高亲和性8,9和类似的活化的血小板表面已经开发了10。 FVIII的连续螺旋机构势必LNT已经被证明是effecti对于已经通过结构冷冻EM 5测定FVIII膜结合状态。官能化LNT是一个理想的系统通过冷冻电镜11，12，以研究蛋白质-蛋白质和螺旋组织膜相关蛋白的蛋白质-膜相互作用。冷冻电镜具有如下优点：与传统的结构的方法，如X-射线晶体学和核磁共振，作为试件被保存在最靠近生理环境（缓冲液，膜，pH值），无添加剂和同位素。在FVIII的情况下，使用此技术研究与膜结合的结构更是生理上相关的，因为在LNT类似密切的大小，形状和组成的活化的血小板，其中Tenase配合装配体内的伪足。 缺陷和FVIII的原因A型血友病，严重的出血性疾病影响1 5000男性的人口4,6的缺乏。在大多数电子ffective治疗血友病A是重组人FVIII（hFVIII的）终身管理。的重组FVIII血友病A治疗的显著并发症是抑制性抗体的发展到人的形式影响血友病A患者13约30％。在这种情况下，猪FVIII（pFVIII）浓缩物的情况下，如猪FVIII显示低交叉反应性抗人FVIII和形式的功能性复合体与人的FIXa 7的抑制性抗体。建立两个猪和人FVIII形式的膜结合组织是重要的，了解的FVIII辅助因子的功能和血止血的影响的结构基础。 在这项研究中，我们描述了脂质纳米技术，冷冻电镜和结构分析，旨在解决两个高度同源的FVIII形式的膜结合组织的结合。所提出的Cryo-EM数据和三维结构螺旋举办porci在带负电荷的LNT NE和人FVIII表明，所提出的纳米技术作为基础，FVIII的结构测定与膜结合的凝血因子和在生理环境中的膜复合物的潜力。

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">JEM2100 with LaB6&#160;</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;">JEM-2100</td>
			<td style="width:447px;">operated at 200 kV</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">with TEMCON software</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan626 Cryo-holder</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">626.DH</td>
			<td style="width:447px;">cooled to -175 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">with temperature controler unit</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan 4K x 4K CCD camera</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">US4000</td>
			<td style="width:447px;">4096 x 4096 pixel at 15 microns/pixel physical resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Solarus Model 950 plasma cleaner</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Vitrobot Mark IV</td>
			<td style="width:205px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Materials</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Carbon coated 300 mesh 3mm copper grid</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">01821</td>
			<td style="width:447px;">plasma cleaned for 10 s on high power</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">Quantifoil R2/2 300 mesh</td>
			<td style="width:205px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">Q225-CR2</td>
			<td style="width:447px;">Carbon coated 300 mesh Cu grids with 2 mm in diameters holes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Uranyl acetate dihydrate</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">19481</td>
			<td style="width:447px;">1% solution, filtered</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:239px;">Galactosyl ceramide</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">860546</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dioleoyl-sn-glycero-phospho-L-serine</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">840035</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Software</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:239px;">EM software Digital Micrograph</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.gatan.com/DM/">http://www.gatan.com/DM/</a></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">EM software EMAN</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://blake.bcm.edu/emanwiki/EMAN/">http://blake.bcm.edu/emanwiki/EMAN/&#160;</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">EM software Spider</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://spider.wadsworth.org/spider_doc/spider/docs/spider.html">http://spider.wadsworth.org/spider_doc/spider/docs/spider.html</a></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EM software IHRSR</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;">Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (IMOD)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (SerialEM)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="ftp://bio3d.colorado.edu/pub/SerialEM/">ftp://bio3d.colorado.edu/pub/SerialEM/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">UCSF-Chimera</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.cgl.ucsf.edu/chimera/download.html">http://www.cgl.ucsf.edu/chimera/download.html</a></td>
		</tr>
	</tbody>
</table>

helical organization of blood coagulation factor viii on lipid nanotubes

<html>低温电子显微镜（冷冻电镜）1是一款功能强大的方法在水合状态和膜环境2，调查的蛋白质和复合物的功能结构。 凝血因子VIII（FVIII） 图3是一个多结构域的血浆糖蛋白。缺陷或FVIII的缺乏是导致血友病A型 - 一个严重的出血性疾病。经蛋白水解活化，FVIII结合到丝氨酸蛋白酶因子IXa上的带负电荷的血小板膜，这是正常的凝血4是至关重要的。尽管举足轻重的作用FVIII凝血发挥，其膜结合态的结构信息是不完整的5。重组FVIII浓缩物是抗血友病A型的最有效的药物和市售的FVIII可以表示为人类或猪，既形成功能性复合物与人的因子IXa 6,7。 “&gt;在本研究中，我们提出低温电子显微镜（冷冻电镜）的组合，脂质纳米技术和结构分析应用到解决两个高度同源的FVIII形式与膜结合的结构：人和猪在我们实验室开发的方法以螺旋状组织两个功能重组FVIII形式对带负电荷的脂质纳米管（LNT）中说明。代表结果表明，我们的方法是足够灵敏定义序列中的两个高度同源（86％序列同一性之间的差异在螺旋机构）蛋白。为螺旋机构，冷冻电镜和电子断层扫描（ET）的数据采集的详细方案中给出的二维（2D）和三维（3D）结构分析应用于获得人和猪的三维重建FVIII-LNT进行了讨论。所提出的人类和猪FVIII-LNT结构表明了该方法的潜力度的计算E中的功能性，膜结合的凝血因子VIII在高分辨率的组织。

重组人类和猪FVIII被成功地组织上的螺旋形带负电荷的单一双层LNT，类似的活化的血小板表面。人类和猪FVIII-LNT的螺旋机构通过所收集的数字显微图是一致的（ 图2）。控制LNT和人类和猪FVIII-LNT螺旋管被选择和分割的e2helixboxer.py GUI和与e2workflow.py GUI，单一粒子的选择（ 表1）中创建的初始数据集。 膜结合人和猪FVIII-LNT的螺旋顺序从傅立叶?...

Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

我们提出了低温电子显微镜，脂质纳米技术和结构分析的组合应用，以解决两个高度同源的FVIII形式与膜结合的结构：人和猪。在我们研制的实验室，以螺旋状组织两个功能重组FVIII形式对带负电荷的脂质纳米管（LNT）的方法进行说明。

凝血因子VIII的脂质纳米管螺旋组织

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Research

JoVE Journal

Bioengineering

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

同时合成单壁碳纳米管和石墨在电弧等离子体磁增强

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

科研

生物工程

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

描述了微RNA转录的转录因子依赖调节

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...