Photoinduced structural changes in the collagen/gelatin binding domain of fibronectin.
Prolonged exposure of human plasma fibronectin (pFn) and its 40- and 21-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. Fluorescence polarization binding assays of TR-CB7 with pFn and the 40-kDa GBF yielded progressively higher Kd's with increased time of exposure to 280-nm light at 25 degrees C. Binding of nonirradiated and irradiated pFn and fragments to gelatin-Sepharose correlated with the polarization 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 conformational changes; the maximum fluorescence wavelength red-shifted from between 340 and 350 nm to 360 nm, with concomitant increases in fluorescence intensity. Exposure of 21- and 40-kDa GBFs and pFn to 280-nm light also generated approximately two, four, and six free sulfhydryl groups 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 affinity for gelatin or the collagen fragment result from structural changes secondary to the breakage of disulfide bonds, as a consequence of energy transfer from nearby tryptophans in one or more of the Fn type I repeats in the gelatin binding region of fibronectin.
Miles
A M
AM
Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport 71130, USA.
Brew
S A
SA
Ingham
K C
KC
Smith
R L
RL
D003094:Collagen / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008027:Light; D011487:Protein Conformation / Q000528:radiation effects; D013050:Spectrometry, Fluorescence
D004220:Disulfides; D005353:Fibronectins; D005780:Gelatin; D003094:Collagen