title: Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices.
abstract: 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 force 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.
authors.affiliation: Biomedical Engineering Program, Department of Anatomy and Cell Biology, Rheumatology Division, Indiana University-Purdue University at Indianapolis, Indiana University School of Medicine, Indiana, Indianapolis 46202, USA.
authors.forename: H B
authors.initials: HB
authors.lastname: Sun
authors.forename: G N
authors.initials: GN
authors.lastname: Smith
authors.forename: K A
authors.initials: KA
authors.lastname: Hasty
authors.forename: H
authors.initials: H
authors.lastname: Yokota
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003094:Collagen / Q000737:chemistry*; D004285:Dogs; D006801:Humans; D020784:Matrix Metalloproteinase 8 / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D003094:Collagen; D020784:Matrix Metalloproteinase 8