Name: a nanobar-supported lipid bilayer system for the study of membrane curvature sensing proteins in vitro
Uploaded: 2022-11-30T13:30:00
Description: Watch this Scientific Journal Video about A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro at JoVE.com

我们感谢南洋理工大学（NTU）南洋纳米制造中心（N2FC）和颠覆性光子技术中心（CDPT）支持纳米结构制造和SEM成像，感谢南洋理工大学生物科学学院蛋白质生产平台（PPP）用于蛋白质纯化，感谢南洋理工大学化学与生物医学工程学院的共聚焦显微镜。这项工作由新加坡教育部（W. Zhao，RG112/20，RG95/21和MOE-T2EP30220-0009），数字分子分析与科学研究所（IDMxS）资助，由教育部资助的卓越研究中心计划（W. Zhao），人类前沿科学计划基金会（W. Zhao，RGY0088 / 2021），NTU启动基金（W. Zhao）， 南洋理工大学化学与生物医学工程学院研究奖学金（苗X.）和中国国家留学基金委研究奖学金（吴杰）。

1. 纳米芯片的清洗
<ol><li>将纳米芯片放入 10 mL 烧杯中，图案面朝上。 注意：该石英纳米芯片已通过电子束光刻 技术制造 ，如前所述 21.芯片上纳米结构的几何形状和排列可以定制设计。这里使用的梯度纳米棒的尺寸为长度为2000 nm，高度为600 nm，宽度为100至1000 nm（步长为100 nm）。</li>
	<li>小心地向烧杯中加入 1 mL 98% 硫酸，并确保酸完全覆盖芯片的正面和?...

<ol>
	<li>McMahon, H. T., Gallop, J. L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature. 438 (7068), 590-596 (2005).</li>
	<li>Zhao, W. et al. Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nature Nanotechnology. 12 (8), 750-756 (2017).</li>
	<li>Galic, M. et al. External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane. Nature Cell Biology. 14 (8), 874-881 (2012).</li>
	<li>Rosholm, K. R. et al. Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells. Nature Chemical Biology. 13 (7), 724-729 (2017).</li>
	<li>Lou, H. Y. et al. Membrane curvature underlies actin reorganization in response to nanoscale surface topography. Proceedings of the National Academy of Sciences. 116 (46), 23143-23151 (2019).</li>
	<li>Cail, R. C., Shirazinejad, C. R., Drubin, D. G. Induced nanoscale membrane curvature bypasses the essential endocytic function of clathrin. Journal of Cell Biology. 221 (7), e202109013 (2022).</li>
	<li>Mu, H. et al. Patterning of oncogenic ras clustering in live cells using vertically aligned nanostructure arrays. Nano Letter. 22 (3), 1007-1016 (2022).</li>
	<li>Peter, B. J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science. 303 (5657), 495-499 (2004).</li>
	<li>Bigay, J., Casella, J. F., Drin, G., Mesmin, B., Antonny, B. ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. The EMBO Journal. 24 (13), 2244-2253 (2005).</li>
	<li>Ebrahimkutty, M. P., Galic, M. Receptor-free signaling at curved cellular membranes. Bioessays. 41 (10), e1900068 (2019).</li>
	<li>Bhatia, V. K. et al. Amphipathic motifs in BAR domains are essential for membrane curvature sensing. The EMBO Journal. 28 (21), 3303-3314 (2009).</li>
	<li>Larsen, J., Hatzakis, N. S., Stamou, D. Observation of inhomogeneity in the lipid composition of individual nanoscale liposomes. Journal of the American Chemical Society. 133 (28), 10685-10687 (2011).</li>
	<li>Prevost, C. et al. IRSp53 senses negative membrane curvature and phase separates along membrane tubules. Nature Communications. 6, 8529 (2015).</li>
	<li>Simunovic, M. et al. How curvature-generating proteins build scaffolds on membrane nanotubes. Proceedings of the National Academy of Sciences. 113 (40), 11226-11231 (2016).</li>
	<li>Holkar, S. S., Kamerkar, S. C., Pucadyil, T. J. Spatial control of epsin-induced clathrin assembly by membrane curvature. Journal of Biological Chemistry. 290 (23), 14267-14276 (2015).</li>
	<li>Dar, S., Kamerkar, S. C., Pucadyil, T. J. Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions. Nature Protocols. 12 (2), 390-400 (2017).</li>
	<li>Nair, P. M., Salaita, K., Petit, R. S., Groves, J. T. Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. Nature Protocols. 6 (4), 523-539 (2011).</li>
	<li>Lee, I. H., Kai, H., Carlson, L. A., Groves, J. T., Hurley, J. H. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly. Proceedings of the National Academy of Sciences. 112 (52), 15892-15897 (2015).</li>
	<li>Beber, A. et al. Membrane reshaping by micrometric curvature sensitive septin filaments. Nature Communications. 10 (1), 420 (2019).</li>
	<li>Bridges, A. A., Jentzsch, M. S., Oakes, P. W., Occhipinti, P., Gladfelter, A. S. Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton. Journal of Cell Biology. 213 (1), 23-32 (2016).</li>
	<li>Li, X. et al. A nanostructure platform for live-cell manipulation of membrane curvature. Nature Protocols. 14 (6), 1772-1802 (2019).</li>
	<li>Su, M. et al. Comparative study of curvature sensing mediated by F-BAR and an intrinsically disordered region of FBP17. iScience. 23 (11), 101712 (2020).</li>
	<li>Mayer, L. D., Hope, M. J., Cullis, P. R. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochimica et Biophysica Acta. 858 (1), 161-168 (1986).</li>
	<li>Santoro, F. et al. Revealing the cell-material interface with nanometer resolution by focused ion beam/scanning electron microscopy. ACS Nano. 11 (8), 8320-8328 (2017).</li>
	<li>Platt, V. et al. Influence of multivalent nitrilotriacetic acid lipid-ligand affinity on the circulation half-life in mice of a liposome-attached His6-protein. Bioconjugate Chemistry. 21 (5), 892-902 (2010).</li>
	<li>Williams, D., Vicogne, J., Zaitseva, I., McLaughlin, S., Pessin, J. E. Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane. Molecular Biology of the Cell. 20 (23), 4910-4919 (2009).</li>
	<li>El Alaoui, F. et al. Structural organization and dynamics of FCHo2 docking on membranes. Elife. 11, e73156 (2022).</li>
	<li>Seu, K. J. et al. Effect of surface treatment on diffusion and domain formation in supported lipid bilayers. Biophysical Journal. 92 (7), 2445-2450 (2007).</li>
	<li>Hung, Y. F. et al. Amino terminal region of dengue virus NS4A cytosolic domain binds to highly curved liposomes. Viruses. 7 (7), 4119-4130 (2015).</li>
	<li>Hatzakis, N. S. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nature Chemical Biology. 5 (11), 835-841 (2009).</li>
	<li>Johnson, J. M., Ha, T., Chu, S., Boxer, S. G. Early steps of supported bilayer formation probed by single vesicle fluorescence assays. Biophysical Journal. 83 (6), 3371-3379 (2002).</li>
	<li>Jing, Y., Trefna, H., Persson, M., Kasemo, B., Svedhem, S. Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size. Soft Matter. 10 (1), 187-195 (2014).</li>
	<li>Cole, R. W., Jinadasa, T., Brown, C. M. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nature Protocols. 6 (12), 1929-1941 (2011).</li>
	<li>Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Developmental Cell. 9 (6), 791-804 (2005).</li>
	<li>Florentsen, C. D. et al. Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles. Soft Matter. 17 (2), 308-318 (2021).</li>
	<li>Sarkar, Y., Majumder, R., Das, S., Ray, A., Parui, P. P. Detection of curvature-radius-dependent interfacial pH/polarity for amphiphilic self-assemblies: positive versus negative curvature. Langmuir. 34 (21), 6271-6284 (2018).</li>
	<li>Raiborg, C., Stenmark, H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature. 458 (7237), 445-452 (2009).</li>
	<li>Alqabandi, M. et al. The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization. BMC Biology. 19 (1), 66 (2021).</li>
	<li>Leitenberger, S. M., Reister-Gottfried, E., Seifert, U. Curvature coupling dependence of membrane protein diffusion coefficients. Langmuir. 24 (4), 1254-1261 (2008).</li>
	<li>Bozelli, J. C., Jr. et al. Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates. Journal of Biological Chemistry. 293 (46), 17780-17791 (2018).</li>
	<li>Parthasarathy, R., Yu, C. H., Groves, J. T. Curvature-modulated phase separation in lipid bilayer membranes. Langmuir. 22 (11), 5095-5099 (2006).</li>
	<li>Yuan, F. et al. Membrane bending by protein phase separation. Proceedings of the National Academy of Sciences. 118 (11), e2017435118 (2021).</li>
	<li>London, E. Membrane structure-function insights from asymmetric lipid vesicles. Accounts of Chemical Research. 52 (8), 2382-2391 (2019).</li>
	<li>Rossetti, F. F., Textor, M., Reviakine, I. Asymmetric distribution of phosphatidyl serine in supported phospholipid bilayers on titanium dioxide. Langmuir. 22 (8), 3467-3473 (2006).</li>
	<li>Richter, R. P., Maury, N., Brisson, A. R. On the effect of the solid support on the interleaflet distribution of lipids in supported lipid bilayers. Langmuir. 21 (1), 299-304 (2005).</li>
	<li>Wacklin, H. P., Thomas, R. K. Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles. Langmuir. 23 (14), 7644-7651 (2007).</li>
	<li>Lin, W. C., Blanchette, C. D., Ratto, T. V., Longo, M. L. Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study. Biophysical Journal. 90 (1), 228-237 (2006).</li>
</ol>

这里描述的纳米棒-SLB系统提供了几种现有体外测定中优势的独特组合。作为脂质体漂浮或沉降测定，它有效地揭示了蛋白质与高度弯曲的膜的优先结合，但需要的样品要少得多，并且在单个纳米棒8，29上提供更精确定义的曲率。它还提供广泛的精确控制曲率，可与SLiC测定同时进行比较，较少关注不同曲率下的脂质组成变化，以及随着尺寸低于光...

膜曲率是细胞的关键物理参数，发生在各种细胞过程中，例如形态发生、细胞分裂和细胞迁移1.现在人们普遍认为，膜曲率不仅仅是细胞事件的简单结果;相反，它已成为蛋白质相互作用和细胞内信号传导的有效调节剂。例如，发现参与网格蛋白介导的内吞作用的几种蛋白质优先与弯曲膜结合，导致内吞作用2的热点形成。膜变形有许多不同的原因，例如细胞骨架力拉动膜，存在不同大小头基的脂质不对称性，存在锥形跨膜蛋白，膜成形蛋白如BAR结构域蛋白（以Bin，两栖蛋白和Rvs蛋白命名）的积累，以及两亲性螺旋结构域插入膜1.有趣的是，这些蛋白质和脂质不仅使膜变形，而且还可以感知膜曲率并表现出优先积累1。因此，研究具有不同曲率的膜是否以及如何改变附着在其上的蛋白质和脂质的分布和动力学以及对相关细胞内过程的潜在影响至关重要。
已经开发了许多技术来分析活细胞和体外系统中弯曲膜和蛋白质之间的相互作用。活细胞系统提供了一个真实的细胞环境，具有丰富的脂质多样性和动态蛋白质信号传导调节2，3，4，5，6，7。然而，由于细胞内环境的不确定性和波动性，这种复杂的系统很难研究。因此，使用由已知脂质种类和纯化蛋白质组成的人造膜的体外测定已成为表征蛋白质和弯曲膜之间关系的强大重构系统。传统上，通过挤出产生不同直径的脂质体，以通过使用离心力的共沉降测定或具有密度梯度的共浮选测定来检测曲率敏感的蛋白质，以避免蛋白质聚集8，9。然而，挤出脂质体的曲率受到挤出机10中使用的膜过滤器的可用孔径的限制。单脂质体曲率（SLiC）测定已被证明可以克服这一限制，其中不同直径的脂质体被荧光标记并固定在表面上，以便曲率可以用荧光强度11标记。然而，在小囊泡中观察到脂质组成的强烈变化，这影响了曲率测量的准确性12。系绳拉动实验使用光学镊子更准确地测量从巨型单层囊泡 （GUV） 拉出的瞬态系绳上的曲率，其中曲率可以通过产生的膜张力很好地控制13，14。该方法适用于研究正曲率或负曲率传感蛋白，但受到管生成10的通量的限制。支持的膜管 （SMrT） 测定可同时生成多个膜管，这些膜管通过微流体流从同一脂质储层挤出。然而，膜曲率沿纳米管固有地变化，这损害了基于荧光强度的曲率测量的准确性15，16。相比之下，使用小的单层囊泡（SUV，直径<100nm17）在含有设计形貌的表面上形成支持的脂质双层（SLB），产生单个双层膜，其曲率由纳米制造或纳米材料预先确定，精度为18，19，20。
在这里，我们提出了一种在具有纳米棒阵列的制造纳米芯片表面上形成SLB的协议，以及如何使用它来探测 体外蛋白质的曲率敏感性。如图 1所示，该测定有六个基本组成部分：A）用微流体室清洁和组装芯片;B）制备具有确定脂质组成的SUV;C）在纳米芯片上形成SLB并与曲率敏感蛋白结合;D）在荧光显微镜下对SLB和曲率敏感蛋白进行成像和表征;e） 清洁芯片以便重复使用;F）用于定量分析蛋白质曲率传感能力的图像处理。下面将逐步介绍详细的协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anhydrous Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Diamond</td>
			<td>RN0879999FU</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber Vial</td>
			<td>Sigma-Aldrich</td>
			<td>27115-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain PS: L-&#945;-phosphatidylserine (Brain, Porcine) (sodium salt)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>840032</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Beaker</td>
			<td>Schott-Duran</td>
			<td>SCOT211060804</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Beaker</td>
			<td>Schott-Duran</td>
			<td>SCOT211061706</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Beaker</td>
			<td>Schott-Duran</td>
			<td>SCOT211065408</td>
			<td>The second container&#160;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>V800117</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td>Watsons</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18:1 DGS-NTA(Ni): 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>790404</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg PC: L-&#945;-phosphatidylcholine (Egg, Chicken)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>840051</td>
			<td></td>
		</tr>
		<tr>
			<td>F-BAR</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>F-BAR+IDR</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>GFP-His</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad</td>
			<td>V9.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide, 30% (Certified ACS)</td>
			<td>Thermo Scientific</td>
			<td>H325-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IDR from human FBP17</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>1.50d</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscopy</td>
			<td>Zeiss&#160;</td>
			<td>LSM 800 with Airyscan</td>
			<td>100x (N.A.1.4) oil objective.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher scientific</td>
			<td>10010240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extuder&#160;</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>610000-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microtubes</td>
			<td>Greiner</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2018b</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclepore Hydrophilic Membrane,0.1 &#956;m</td>
			<td>Whatman</td>
			<td>800309</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufferen Saline (PBS)</td>
			<td>Life Technologies Holdings Pte Ltd.</td>
			<td>70013</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Base</td>
			<td>Dow Corning Corporation</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Crosslinker</td>
			<td>Dow Corning Corporation</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>HARRICK PLASMA</td>
			<td>PDC-002-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz Nanochip</td>
			<td>Donghai County Alfa Quartz Products CO., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>795429</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red DHPE: Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt</td>
			<td>Life Technologies Holdings Pte Ltd.</td>
			<td>T1395MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Gooi</td>
			<td>PDC-002-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaners</td>
			<td>Elma</td>
			<td>D-78224</td>
			<td></td>
		</tr>
		<tr>
			<td>Voterx</td>
			<td>Scientific Industries</td>
			<td>G560E</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>NUCERITE</td>
			<td>5312</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Julabo</td>
			<td>TW8</td>
			<td></td>
		</tr>
	</tbody>
</table>

a nanobar-supported lipid bilayer system for the study of membrane curvature sensing proteins in vitro

膜曲率在细胞的各种基本过程中起着重要作用，例如细胞迁移、细胞分裂和囊泡运输。它不仅由细胞活动被动产生，而且还主动调节蛋白质相互作用并参与许多细胞内信号传导。因此，研究膜曲率在调节蛋白质和脂质的分布和动力学中的作用具有重要价值。最近，已经开发了许多技术来研究体 外弯曲膜和蛋白质之间的关系。与传统技术相比，新开发的纳米巴支持的脂质双层（SLB）通过在具有预定义膜曲率和局部平坦控制的纳米条图案阵列上形成连续的脂质双层，在膜曲率生成方面提供了高通量和更高的精度。脂质流动性和蛋白质对弯曲膜的敏感性都可以使用荧光显微镜成像进行定量表征。本文介绍了如何在含有纳米棒阵列的人造玻璃表面上形成SLB的详细程序，以及如何在这种SLB上表征曲率敏感蛋白。此外，还涵盖了纳米芯片重用和图像处理的协议。除了纳米巴-SLB，该协议还适用于用于曲率传感研究的所有类型的纳米结构玻璃芯片。

建议使用纳米棒设计来探测正曲率传感蛋白，其两端包含一个半圆，曲率由纳米条宽度定义，中心局部有一个平坦/零曲率控制（图 2A，B）。在纳米棒上成功形成SLB会导致脂质标记信号均匀分布在整个纳米棒表面上，如图 2C所示。来自多个纳米棒的信号可以通过平均单个纳米棒图像来组合（图2D），以便可以最小化不?...

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A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

在这里，开发了一种纳米巴支持的脂质双层系统，以提供具有定义曲率的合成膜，从而能够在 体外表征具有曲率传感能力的蛋白质。

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

这种纳米棒支持的脂质双层系统可用于研究膜曲率在调节细胞活动期间蛋白质和脂质的动力学和分布中的作用。该技术通过在专利纳米巴误差上形成连续的脂质双层，通过高分辨率纳米制造预定义的膜曲率，在膜曲率生成中提供高亚根和不良。首先，将纳米芯片放入 10 毫升烧杯中，图案面朝上。小心地在烧杯中加入一毫升98%的硫酸，并确保酸完全覆盖芯片的正面和背面。慢慢旋转烧杯，一滴一滴地加入 200 微升 30% 过氧化氢，直到整个烧杯变热。确保硫酸和过氧化氢充分混合形成食人鱼溶液，用于从纳米芯片中去除有机分子。将烧杯放入二次玻璃容器中，并将纳米芯片浸入食人鱼溶液中过夜，以彻底清洁杂质。取出烧杯，小心地将食人鱼溶液移入酸性废物容器中。将五毫升去离子水装入烧杯中以稀释残留的酸并将其丢弃到酸性废物中。重复此步骤五次。用镊子抓住芯片，用连续的去离子水流清洗，彻底去除残留的酸。用99.9%的氮气吹干芯片以形成SLB。超声处理脂质混合物30分钟。将脂质混合物在液氮中冷冻20秒，然后在水浴中以42摄氏度解冻两分钟。重复冻融循环30次。之后，脂质混合物看起来像透明液体。将脂质混合物通过小型挤出机并来回挤出20次。从另一侧的注射器中收集SUV，以减少较大颗粒或异物的污染，并将其转移到1.5毫升离心管中。用镊子小心地从去离子水中取出清洁的纳米芯片，并用99.9%的氮气吹干。用空气等离子处理对纳米芯片进行一小时的表面清洁。将纳米芯片组装在PDMS室中。首先将芯片放在干净的表面上，图案朝上。用芯片轻轻覆盖中间的PDMS，并确保整个图案暴露在中间PDMS中椭圆形大开口的中心区域。用中间的PDMS覆盖顶部的PDMS，并将其两个小孔保留在中间PDMS的大椭圆孔区域内。然后，组装PDMS室。用移液管将SUV从顶部PDMS上的两个小孔之一装入PDMS通道，并在室温下孵育15分钟以形成SLB。从小孔的一侧轻轻地将PBS缓冲液移入PDMS通道，然后用棉签从另一个孔中清除废物，以洗掉未绑定的SUV。然后获取在纳米芯片上形成的SLB。使用100 x油物镜设置激光扫描共聚焦显微镜。打开Zen软件，选择可以激发脂质和蛋白质荧光的激发激光功率。选择采集模式。然后单击智能设置，然后单击德州红。使用对焦旋钮调整焦点以定位芯片上的纳米条，直到纳米条边缘在脂质通道下方清晰。在添加蛋白质之前，设置扫描参数以获得脂质通道的控制图像。通过在随机的单个纳米巴区域上用荧光标记的脂质双层漂白来进行 FRAP 测定。选中“实验区域”和“漂白”复选框。画一个直径为五微米的圆形区域，其中可以包括中心的整个纳米棒，并添加到实验区域。输入延时摄影成像参数。选择时间序列。然后单击持续时间。选择 100 个周期，间隔等于 2 秒。输入漂白参数。选中执行 FRAP 的激光复选框并将功率更改为 100%单击开始实验以进行 FRAP 实验。将蛋白质溶液加载到PDMS通道中，并在室温下孵育五分钟，以使蛋白质与SLB结合。重新聚焦纳米棒并设置扫描参数，以拍摄脂质和蛋白质通道的图像，以进行蛋白质曲率传感检测。选择实验区域并在脂质和蛋白质通道上进行FRAP测定，并进行延时成像以表征曲率传感蛋白的迁移率。两者都显示出在SLB涂层的单个纳米棒末端的积累增加，宽度为300纳米。在这里，SLB含有10%PS以静电增强蛋白质结合。这两种蛋白质的端中心比脂质高，约为1。在比较 IDR FBP17 和 F-Bar 时，IDR FBP17 的端中心比表明比 F-Bar 更强的曲率感应。当曲率减小到直径400纳米以下时，所有3种蛋白质在纳米条末端弯曲膜位点均表现出优先积累。在这三个蛋白质结构域中，IDR FBP17给出的纳米条和密度最高，表明其最强的曲率传感能力，而F-Bar显示出最低的值。通过逐渐增加蛋白质浓度可以绘制蛋白质结合曲线，表明IDR FBP17具有很强的协同曲率传感能力。与纳米棒上脂质信号的快速恢复相比，F-Bar无法在两分钟内恢复，这表明其在弯曲膜部位的膜迁移率和缔合动态显着降低。令人惊讶的是，与F-Bar的行为不同，IDR FBP17信号在同一时间范围内表现出明显的恢复，表明IDR FBP17在弯曲膜上积累的动态性质。然而，在洗去溶液中未结合的IDR FBP17后，无法观察到与以前相同的IDR FBP17通道恢复。在研究弯曲膜和蛋白质之间的相互作用时，SLB质量非常重要。因此，在我们的实验中，FRAP测试对于验证膜迁移率是必要的。该系统将帮助研究人员研究影响蛋白质与弯曲膜相互作用的各种参数，例如曲率传感域和脂质组成，以及对弯曲膜进行动态研究，例如面部分离行为。

Research

JoVE Journal

Biochemistry

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...