调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统
Summary
Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.
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。
Introduction
Cellulosomes are large multienzyme complexes displayed on the surface of anaerobic cellulolytic bacteria (e.g. C. thermocellum) that have evolved to efficiently depolymerize plant cell wall lignocellulose into soluble oligosaccharides1. A central attribute of cellulosomes is the high-affinity cohesin-dockerin interaction. In the most prominent paradigm, a highly conserved 60-75 amino acid type I dockerin module is displayed at the C-terminal end of the various bacterial enzymes. The dockerin module directs assembly of synergistic combinations of enzymes onto the noncatalytic scaffold protein ('scaffoldin'), which comprises a polyprotein of cohesin domains that are specific for the type I dockerin module. At higher levels, cellulosome architecture can become very complex, incorporating alternative cohesin and dockerin pairs (e.g. type II, type III) that anchor the structures to the cell surface and allow for the assembly of branched structures containing multiple scaffoldins2. The various cohesin-dockerin types, despite having related structures, exhibit differential binding specificities suppressing cross reactivity with unintended scaffoldins or components from other cellulosome-producing bacterial species. While bioinformatic approaches have successfully identified thousands of unique cellulosomal components at the genetic level, comparatively few protein structures are known, and the mechanisms at work in cohesin-dockerin specificity determination remains an active area of structural biology research.
Since the invention of the atomic force microscope (AFM) by Binnig et al.3, various AFM operational modes have been developed and continuously improved, including noncontact imaging, oscillation mode imaging4, and single molecule force spectroscopy (SMFS)5,6. SMFS has evolved into a widely used technique to directly probe individual proteins7-11, nucleic acids12-15, and synthetic polymers16-19. In a typical SMFS experiment to investigate receptor-ligand binding20,21, an AFM cantilever tip is modified with one of the binding partners, while a flat glass surface is modified with the complementary binding partner. The modified cantilever is brought into contact with the surface allowing the partners to bind. The base of the cantilever is then withdrawn at constant speed and the force is measured using the optical lever deflection method. The resultant force-distance data traces exhibit sawtooth-like peaks if binding was established. In cases where the binding partners are fused to multiple protein domains, each peak in the force-distance trace can be correlated to the unfolding of a single protein domain or folded subdomain, while the last peak corresponds to rupture of the protein binding interface. The specific positions of the force-resistant elements can be used as a fingerprint to identify the various protein domains of interest. This method can be used to interrogate important amino acids involved in protein folding and stabilization. Many models have been reported in the literature to treat the characteristic force extension behavior observed in SMFS experiments. The most commonly used models include the freely jointed chain (FJC) model22, the worm-like chain (WLC) model18,23-25, and the freely rotating chain (FRC) model25,26.
In our prior work11, we used single-molecule force spectroscopy to investigate the interaction of cohesin and dockerin modules. Here, we present an experimental protocol for glass surface and cantilever functionalization with enzyme-dockerin and CBM-cohesin protein constructs. We also present an AFM-based SMFS protocol including data acquisition and analysis procedures. The described protocol can easily be generalized to other molecular systems, and should prove particularly useful to researchers interested in high-affinity receptor ligand pairs.
Protocol
Representative Results
Discussion
To obtain meaningful data from single molecule force spectroscopy experiments, it is crucial to achieve well-defined and reproducible pulling geometries. The protocol used here results in site-specific immobilization of protein complexes in a defined pulling geometry.
The cantilevers used in this study were chosen due to their force sensitivity and high resonance frequency in water. Moreover, the small tip curvature of approximately 10 nm is advantageous for single molecule experiments due to …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding from a European Research Council advanced grant to Hermann Gaub. Michael A. Nash gratefully acknowledges funding from Society in Science – The Branco Weiss Fellowship program. The authors thank Edward A. Bayer, Yoav Barak, and Daniel B. Fried at the Weizmann Institute of Science for generously providing the proteins used in this study. The authors thank Hermann E. Gaub, Elias M. Puchner, and Stefan W. Stahl for helpful discussions.
Materials
| 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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