Dissociation kinetics of an enzyme-inhibitor system using single-molecule force measurements.
We report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme-inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s(-1) for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.
Department of Physics and Astronomy, Wayne State University, Detroit, Michigan 48201, United States.
Essa
E
Mayyas
Margarida
M
Bernardo
Lindsay
L
Runyan
Anjum
A
Sohail
Venkatesh
V
Subba-Rao
Mircea
M
Pantea
Rafael
R
Fridman
Peter M
PM
Hoffmann
D004791:Enzyme Inhibitors / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D008722:Methods; D018625:Microscopy, Atomic Force / Q000379:methods*; D009362:Neoplasm Metastasis; D011485:Protein Binding; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D019715:Tissue Inhibitor of Metalloproteinase-1; C120487:pro-matrix metalloproteinase 9; D020780:Matrix Metalloproteinase 9