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Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices
Sun, HB
Smith, GN
Hasty, KA
Yokota, H
Analytical Biochemistry, 2000, 283(2): 153-158.
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.
type II collagen; matrix metalloproteinase; MMP-8; atomic force microscopy
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