title: Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope.
abstract: 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 by Clostridium histolyticum collagenases 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.
authors.affiliation: Neuroscience Research Institute, Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara 93106, USA. h_lin@lifesci.ucsb.edu
authors.forename: H
authors.initials: H
authors.lastname: Lin
authors.forename: D O
authors.initials: DO
authors.lastname: Clegg
authors.forename: R
authors.initials: R
authors.lastname: Lal
mesh_terms: D000538:Aluminum Silicates; D000818:Animals; D002118:Calcium / Q000737:chemistry; D003013:Clostridium / Q000201:enzymology; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007091:Image Processing, Computer-Assisted; D061965:Matrix Metalloproteinase Inhibitors; D018625:Microscopy, Atomic Force / Q000379:methods; D051381:Rats; D013499:Surface Properties; D013997:Time Factors
chemical_list: D000538:Aluminum Silicates; D061965:Matrix Metalloproteinase Inhibitors; D003094:Collagen; D004492:Edetic Acid; D017364:Collagenases; D002118:Calcium; C011934:mica