Imaging and mapping heparin-binding sites on single fibronectin molecules with atomic force microscopy.
Fibronectin is composed of multiple homologous repeats and contains many functional domains. Two major heparin-binding domains have previously been identified: the Hep I site near the amino terminus and the Hep II site near the carboxyl terminus. The Hep II site has been considered the high-affinity heparin-binding site based on studies of fibronectin fragments. However, few studies have been carried out on heparin binding by intact fibronectin. We imaged single fibronectin molecules as well as heparin-coated gold particles bound to whole dimeric plasma fibronectin molecules with tapping mode atomic force microscopy. We observed heparin-gold particles preferentially bound at two locations that correspond to the Hep I and Hep II sites. Quantitative analysis of images of individual fibronectin-heparin-gold complexes showed that almost twice as many heparin-gold particles bound to the N-terminal Hep I site compared to the Hep II site. In contrast to previous findings with fibronectin fragments, these results suggest that the Hep I site has a binding affinity higher than or comparable to the Hep II site in the intact fibronectin molecule.
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Neuroscience Research Institute and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106, USA. h_lin@lifesci.ucsb.edu
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D005353:Fibronectins / Q000737:chemistry* / Q000648:ultrastructure; D017979:Gold Colloid / Q000737:chemistry; D006493:Heparin / Q000737:chemistry* / Q000648:ultrastructure; D007089:Image Enhancement; D018625:Microscopy, Atomic Force; D010446:Peptide Fragments / Q000737:chemistry / Q000648:ultrastructure; D010449:Peptide Mapping; D011897:Random Allocation; D012709:Serum Albumin / Q000737:chemistry / Q000648:ultrastructure
D004220:Disulfides; D005353:Fibronectins; D017979:Gold Colloid; D010446:Peptide Fragments; D012709:Serum Albumin; D006493:Heparin