Collagen-protein interactions mapped by phototriggered thiol introduction.

Accumulating evidence shows that collagen plays important roles in many biological systems by interacting with various proteins. The major limitation to determining protein-binding sites on collagen has been a lack of useful methods. We have developed a new strategy for mapping these sites using a photoreactive cross-linker, APDP. A unique -SH group can be introduced into collagen in the vicinity of the protein-binding sites by DTT reduction of the SS bond in the cross-linked product. To identify the sites of cross-linking in collagen, the strategy used was as follows: (i) derivatization of the free -SH group with fluorescein-5-maleimide (FM); (ii) fragmentation of collagen with an appropriate collagenase; (iii) separation of FM-labeled fragments by two-dimensional diagonal electrophoresis; and (iv) detection of cross-linked partners of collagen-binding protein as fluorescent spots under UV light. This strategy can be used to determine the binding sites of various proteins on collagen.