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.
Yasui
Norihisa
N
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan.
Koide
Takaki
T
D001665:Binding Sites; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004164:Diphosphonates / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors; D010449:Peptide Mapping / Q000379:methods*; D010777:Photochemistry; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D000099174:Pigment Epithelium-Derived Factor
D003094:Collagen; D003432:Cross-Linking Reagents; D003545:Cysteine; D004164:Diphosphonates; D005136:Eye Proteins; D020781:Matrix Metalloproteinase 1; D009414:Nerve Growth Factors; D011506:Proteins; D015843:Serpins; D013438:Sulfhydryl Compounds; D000099174:Pigment Epithelium-Derived Factor; C031876:azacycloheptane-2,2-diphosphonate