Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices

作者：Sun HB; Smith GN; Hasty KA; Yokota H^*

来源：Analytical Biochemistry, 2000, 283(2): 153-158.

DOI：10.1006/abio.2000.4629

• type II collagen
• matrix metalloproteinase
• MMP-8
• atomic force microscopy

Type II tropocollagen molecules were reacted with matrix metalloproteinase 8 (MMP-8) and the binding sites as well as the cleavage site of MMP-8 were detected on individual molecules using atomic farce microscopy (AFM). Approximately 300-nm-long coiled-coil tropocollagen molecules were straightened and immobilized on an atomically flat surface for detection by AFM. The direct visualization of individual collagen molecules revealed heterogeneous characteristics of MMP-8 collagen complexes. We observed that there existed multiple MMP-8 nonspecific binding sites on the collagen molecules, but cleavage always took place at a unique site. When collagen molecules, straightened and immobilized on the surface, were reacted with MMP-8, a site of cleavage appeared as a gap in stretched molecules. This is the first report to visually show direct collagenase collagen interactions using AFM. The described AFM-based analysis has potential as a protein analysis tool for understanding a complex mechanism of enzyme:substrate interactions.

• 出版日期2000-8-1