当前位置：
X-MOL 学术
›
Biochemistry
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
A Flow-Extension Tethered Particle Motion Assay for Single-Molecule Proteolysis.
Biochemistry ( IF 2.9 ) Pub Date : 2019-04-12 , DOI: 10.1021/acs.biochem.9b00106 Andrew A Drabek 1 , Joseph J Loparo 1 , Stephen C Blacklow 1
Biochemistry ( IF 2.9 ) Pub Date : 2019-04-12 , DOI: 10.1021/acs.biochem.9b00106 Andrew A Drabek 1 , Joseph J Loparo 1 , Stephen C Blacklow 1
Affiliation
|
Regulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads not be restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow-extension strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allow assignment of tethered substrates to singly captured and multiply tethered bins, with the fraction of fully mobile, single-tether substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiple-tether beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.
中文翻译：
用于单分子蛋白水解的流动延伸系留粒子运动测定。
机械张力下信号蛋白的受调节蛋白水解使细胞能够在各种发育和生理背景下与其环境进行通信。已经使用磁力镊子通过珠系试验在单分子水平上探索了力在诱导蛋白水解敏感性中的作用，但这种努力受到了确保珠子不受多个系绳限制的挑战的限制。在这里，我们描述了一种单分子蛋白水解的多重测定，该测定在配备磁力镊子的显微镜上使用流动延伸策略克服了多重系链问题。流引起的位移的粒子跟踪和计算排序允许将系留基底分配给单独捕获和倍增系留箱，完全移动的单系留基底的分数与盖玻片上加载的基底的浓度成反比。多系链珠的计算排除能够对高度特异性的烟草蚀刻病毒蛋白酶和更混杂的金属蛋白酶 ADAM17 的目标蛋白水解进行可靠的评估。该方法应该普遍适用于多种蛋白酶，并且易于扩展以对作为所施加磁力的函数的蛋白水解敏感性进行稳健评估。
更新日期：2019-04-04
中文翻译：
用于单分子蛋白水解的流动延伸系留粒子运动测定。
机械张力下信号蛋白的受调节蛋白水解使细胞能够在各种发育和生理背景下与其环境进行通信。已经使用磁力镊子通过珠系试验在单分子水平上探索了力在诱导蛋白水解敏感性中的作用，但这种努力受到了确保珠子不受多个系绳限制的挑战的限制。在这里，我们描述了一种单分子蛋白水解的多重测定，该测定在配备磁力镊子的显微镜上使用流动延伸策略克服了多重系链问题。流引起的位移的粒子跟踪和计算排序允许将系留基底分配给单独捕获和倍增系留箱，完全移动的单系留基底的分数与盖玻片上加载的基底的浓度成反比。多系链珠的计算排除能够对高度特异性的烟草蚀刻病毒蛋白酶和更混杂的金属蛋白酶 ADAM17 的目标蛋白水解进行可靠的评估。该方法应该普遍适用于多种蛋白酶，并且易于扩展以对作为所施加磁力的函数的蛋白水解敏感性进行稳健评估。
京公网安备 11010802027423号