单分子检测法在蛋白酶敏感性分析中的应用：力诱导胶原的不稳定性。
Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Collagen Destabilization.
机构信息
Department of Molecular Biology and Biochemistry, Burnaby, British Columbia, Canada.
Department of Molecular Biology and Biochemistry, Burnaby, British Columbia, Canada; Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada.
出版信息
Biophys J. 2018 Feb 6;114(3):570-576. doi: 10.1016/j.bpj.2017.12.006.
Abstract
Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.
摘要
力在调节生物分子结构和相互作用的动力学中起着关键作用，但缺乏技术手段来进行高通量的操纵和连续读出这种响应。我们提出了一种用于测量结构力依赖性可及性的酶促分析方法，该方法利用无线微型离心力显微镜提供实时动力学读数。该显微镜设计简单易用，适合标准的离心机桶，并提供高通量、视频速率的单个蛋白水解切割事件的读出。对数千个连接的胶原蛋白分子的蛋白水解测量显示出负载增强的胰蛋白酶敏感性，表明三螺旋的不稳定性。
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Nat Commun. 2017 Mar 22;8:14913. doi: 10.1038/ncomms14913.
2
Cryptic epitopes and functional diversity in extracellular proteins.细胞外蛋白质中的隐蔽表位与功能多样性
Int J Biochem Cell Biol. 2016 Dec;81(Pt A):112-120. doi: 10.1016/j.biocel.2016.10.020. Epub 2016 Oct 27.
3
Intact Telopeptides Enhance Interactions between Collagens.完整的端肽增强胶原蛋白之间的相互作用。
Biophys J. 2016 Dec 6;111(11):2404-2416. doi: 10.1016/j.bpj.2016.10.039.
4
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5
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Nat Commun. 2016 Mar 17;7:11026. doi: 10.1038/ncomms11026.
6
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7
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8
Effect of intrinsic and extrinsic factors on the simulated D-band length of type I collagen.内在和外在因素对I型胶原蛋白模拟D带长度的影响。
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