Multiplexed single-molecule force proteolysis measurements using magnetic tweezers.
Multiplexed single-molecule force proteolysis measurements using magnetic tweezers.
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DOI：
10.3791/3520
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发表时间：
2012-07
期刊：
影响因子：
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通讯作者：
A. Adhikari;J. Chai;A. Dunn
中科院分区：
文献类型：
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作者：
A. Adhikari;J. Chai;A. Dunn
机械力的产生和检测是细胞生理学的一个普遍存在的方面，与癌症转移（1）、动脉粥样硬化（2）和伤口愈合（3）直接相关。在这些实施例中的每一个中，细胞都对其周围环境施加力，同时酶促重塑细胞外基质（ECM）。力对ECM的影响由于其可能的生物学和医学重要性而成为相当感兴趣的领域（4-7）。单分子技术，如光学捕获（8），原子力显微镜（9）和磁镊（10，11），使研究人员能够通过对单个蛋白质施加力来探测分子水平上酶的功能。在这些技术中，磁镊（MT）以其低成本和高通量而闻名。MT施加的力在~1-100 pN的范围内，并且可以提供毫秒级的时间分辨率，这些品质与单分子水平上的酶机制研究非常匹配（12）。在这里，我们报告了一个高度平行MT测定研究的影响力的蛋白水解的单个蛋白质分子。我们提出了一个三聚体胶原蛋白肽的基质金属蛋白酶1（MMP-1）的蛋白水解的具体例子，然而，这种测定可以很容易地适应于研究其他底物和蛋白酶。
The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis(1), atherogenesis(2) and wound healing(3). In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance(4-7). Single molecule techniques such as optical trapping(8), atomic force microscopy(9), and magnetic tweezers(10,11) allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level(12). Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.