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Bioengineering

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

doi: 10.3791/50950 Published: December 20, 2013
* These authors contributed equally

Abstract

纤维素酶是使用厌氧细菌和真菌的一个子集来消化木质纤维素底物的离散多酶复合物。酶到非催化支架蛋白的大会是由相互作用执导了一系列相关的受体 - 配体对包括互动黏合和锚定模块中。黏合和锚定模块产生解离常数在低皮摩尔到纳摩尔范围,这可能会妨碍精确的解离速率测量结果与传统的大宗方法之间的极强的约束力。单分子力谱(SMFS)与原子力显微镜测量单个生物分子给力的响应,并且相对于其他单分子操纵方法( 光镊),是最佳的学习高亲和力受体-配体相互作用,因为其探测高力政权(> 120 PN)的能力。在这里,我们提出我们的完整协议,用于研究蜂窝织炎osomal蛋白质组件在单分子水平。使用从原生纤维素酶体来源的蛋白质的拓扑结构,我们曾与酶锚定和碳水化合物结合模块黏合(CBM-黏合)融合蛋白,每一个可访问的游离巯基在一个精心设计的半胱氨酸残基。我们提出我们的站点特定的表面固定的协议,以及我们的测量和数据分析程序获得的高亲和力复杂细节的绑定参数。我们将演示如何量化单一子域展开的力量,复杂的破裂力,动力学解离速率,并结合以及潜在的宽度。在表征负责多领域纤维素复合物组装的黏结锚定的相互作用这些方法的成功应用进一步说明。

Materials

Name Company Catalog Number Comments
3-Aminopropyl dimethyl ethoxysilane ABCR GmbH AB110423
5 kDa NHS-PEG-maleimide Rapp Polymer 13 5000-65-35
TCEP Disulfide reducing gel Thermo Scientific, Pierce 77712 www.thermoscientific.com/pierce
Tris(hydroxymethyl)aminomethane
BioLever mini silicon nitride cantilevers Olympus BL-AC40TS-C2 Soft batches
XYZ Piezoelectric actuators Physik Instrumente GmbH
Infrared “broad spectrum” IR laser Superlum
MFP-3D AFM Controller Asylum Research
Igor Pro 6.31 Wavemetrics Data acquisition and analysis
Sodium chloride
Calcium chloride
pH Meter
Sodium borate
Tweezers
Cover glasses Thermo Scientific, Menzel-Gläser 24 mm diameter, 0.5 mm thickness
PTFE sample holder custom made
Sonicator bath
Ethanol analytical purity
Sulfuric acid (concentrated) analytical purity
Hydrogen peroxide (30%) analytical purity
Orbital shaker
Toluene analytical purity
Filter paper
Glass slides
Microtubes
Micropipettes
Centrifuge suitable for microtubes
Rotator
Petri dishes
Beakers
Optical microscope

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调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统
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Cite this Article

Jobst, M. A., Schoeler, C., Malinowska, K., Nash, M. A. Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy. J. Vis. Exp. (82), e50950, doi:10.3791/50950 (2013).More

Jobst, M. A., Schoeler, C., Malinowska, K., Nash, M. A. Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy. J. Vis. Exp. (82), e50950, doi:10.3791/50950 (2013).

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