Stawikowski Maciej J, Knapinska Anna M, Fields Gregg B
Department of Chemistry and Biochemistry, Florida Atlantic University, Jupiter, FL, 33458, USA.
Department of Chemistry, The Scripps Research Institute/Scripps Florida, Jupiter, FL, 33458, USA.
Methods Mol Biol. 2017;1579:137-183. doi: 10.1007/978-1-4939-6863-3_8.
A continuous assay method, such as the one that utilizes an increase in fluorescence upon hydrolysis, allows for rapid and convenient kinetic evaluation of proteases. To better understand MMP behaviors toward native substrates, a variety of fluorescence resonance energy transfer (FRET)/intramolecular fluorescence energy transfer (IFET) triple-helical substrates have been constructed to examine the collagenolytic activity of MMP family members. Results of these studies have been valuable for providing insights into (a) the relative triple-helical peptidase activities of the various collagenolytic MMPs, (b) the collagen preferences of these MMPs, and (c) the relative roles of MMP domains and specific residues in efficient collagenolysis. The present chapter provides an overview of MMP FRET triple-helical substrates and describes how to construct and utilize these substrates.
一种连续测定方法，例如利用水解时荧光增强的方法，可实现对蛋白酶的快速便捷的动力学评估。为了更好地理解基质金属蛋白酶（MMP）对天然底物的作用，人们构建了多种荧光共振能量转移（FRET）/分子内荧光能量转移（IFET）三螺旋底物，以检测MMP家族成员的胶原酶活性。这些研究结果对于深入了解以下方面具有重要价值：（a）各种胶原olytic MMP的相对三螺旋肽酶活性；（b）这些MMP对胶原蛋白的偏好；（c）MMP结构域和特定残基在有效胶原降解中的相对作用。本章概述了MMP FRET三螺旋底物，并描述了如何构建和使用这些底物。
