Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope.
Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope.
复制标题
使用原子力显微镜对单个 I 型胶原蛋白分子的实时蛋白水解进行成像。
DOI：
10.1021/bi990800q
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Lal,R
中科院分区：
文献类型：
--
作者：
Lin,H;Clegg,DO;Lal,R
细胞外基质分子（包括各种胶原蛋白）的合成和降解的动态过程在正常生理功能和病理条件中是重要的。现有的胶原酶降解反应模型来自批量生化测定。在这项研究中，我们已经成像实时个人胶原蛋白I分子和他们的蛋白水解梭菌溶组织胶原酶在磷酸盐缓冲液（PBS）与原子力显微镜（AFM）。我们还成像了胶原酶分子与单个三螺旋胶原蛋白I分子的可能结合和未结合以及胶原蛋白分子亚群的后续蛋白水解。胶原分子的蛋白水解被钙离子减少和酸化所抑制。胶原蛋白水解的AFM研究结果与SDS-PAGE生化测定结果一致。单个胶原蛋白I分子的实时蛋白水解遵循先前从批量生化测定中获得的简单Michaelis-Menton动力学。这是第一个报告的成像实时蛋白水解的单个大分子和其抑制在分子尺度上。单一胶原蛋白分子的蛋白水解动力学与来自批量生化测定的蛋白水解动力学之间的强对应性将在检查实时酶促反应及其在单分子结构水平上的调节方面具有广泛的适用性。这种单分子蛋白质水解的实时研究可以更好地理解蛋白酶与靶蛋白以及蛋白酶与蛋白酶抑制剂之间的相互作用。
The dynamic process of synthesis and degradation of extracellular matrix molecules, including various collagens, is important in normal physiological functions and pathological conditions. Existing models of collagen enzymatic degradation reactions are derived from bulk biochemical assays. In this study, we have imaged in real-time individual collagen I molecules and their proteolysis byClostridium histolyticumcollagenases in phosphate-buffered saline (PBS) with atomic force microscopy (AFM). We have also imaged the likely binding and unbinding of collagenase molecules to single triple-helical collagen I molecules and subsequent proteolysis of subsets of the collagen molecules. The proteolysis of collagen molecules was inhibited by reduced calcium and acidification. Results from AFM study of collagen proteolysis are consistent with SDS−PAGE biochemical assays. The real-time proteolysis of single collagen I molecules followed simple Michaelis-Menton kinetics previously derived from bulk biochemical assays. This is the first report of imaging real-time proteolysis of single macromolecules and its inhibition on a molecular scale. A strong correspondence between the kinetics of proteolysis of single collagen molecules and the kinetics of proteolysis derived from bulk biochemical assays will have a wide applicability in examining real-time enzymatic reactions and their regulation at single molecule structural level. Such real-time study of single molecule proteolysis could provide a better understanding of the interactions between proteases and target proteins as well as proteases and protease inhibitors.