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（ 即光镊），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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