PHOTOINDUCED STRUCTURAL-CHANGES IN THE COLLAGEN GELATIN-BINDING DOMAIN OF FIBRONECTIN

Prolonged exposure of human plasma fibronectin (pFn) and its 40- and 2 1-kDa collagen/ gelatin binding fragments (GBFs) to 280-nm irradiation decreased their affinity for gelatin and for TR-CB7, a fluorescently labeled CNBr fragment of the alpha-1 chain of type I collagen. Fluores cence polarization binding assays of TR-CB7 with pFn and the 40-kDa GB F yielded progressively higher K-d's with increased time of exposure t o 280-nm light at 25 degrees C. Binding of nonirradiated and irradiate d pFn and fragments to gelatin-Sepharose correlated with the polarizat ion data, confirming diminished gelatin binding following exposure to 280-nm light. Fluorescence spectra of intrinsic tryptophans in the 21- and 40-kDa GBFs exhibited changes indicative of photoinduced conforma tional changes; the maximum fluorescence wavelength red-shifted from b etween 340 and 350 nm to 360 nm, with concomitant increases in fluores cence intensity. Exposure of 21- and 40-kDa GBFs and pFn to 280-nm Lig ht also generated approximately two, four, and six free sulfhydryl gro ups per molecule, respectively. No sulfhydryl release was observed in other Trp- and disulfide-containing proteins under the same conditions . We propose that the fluorescence changes as well as the changes in a ffinity for gelatin or the collagen fragment result from structural ch anges secondary to the breakage of disulfide bonds, as a consequence o f energy transfer from nearby tryptophans in one or more of the Fn typ e I repeats in the gelatin binding region of fibronectin.