Evidence for a conformational change in human fibronectin which occurs between 4 and 37 degrees C.
The effects of temperatures between 4 and 37 degrees C on the structure of human fibronectin (Fn) using monoclonal antibodies (MAb) with specificity for Fn have been studied. Three MAb bound to Fn in solution better at 4 than at 37 degrees C, assessed by both ELISA and immunoprecipitation. These MAb detected temp-dependent conformational changes in Fn which were not associated with a major refolding of the Fn molecule, since the S20,w of Fn and the binding of Fn to polyclonal anti-Fn were unchanged by temp shifts from 4 to 37 degrees C. Fn also was adhered directly to ELISA plates at 4 and at 37 degrees C, and then probed with MAb, with detergents, and with trypsin. One MAb bound better to surface adherent Fn when the Fn had bound to the surface at 4 rather than 37 degrees C. Both SDS and polyoxyethylene (20) sorbitan monolaurate (Tween-20) caused the release of more adherent Fn from surfaces to which it had adhered at 4 degrees C. Finally, trypsin released Fn fragments more rapidly and more completely from surfaces coated at 4 than at 37 degrees C. It is concluded that Fn has different conformations at 4 and 37 degrees C in solution and after adherence to surfaces, as reflected by MAb binding, detergent sensitivity and protease susceptibility. This conformational difference is not associated with alterations in the physiochemical properties of the molecule and is thus a result of a less extensive intramolecular rearrangement than the conformational changes resulting from manipulations of ionic strength or pH, and from binding to collagen or hydrophobic surfaces. Nonetheless, the more subtle conformational change demonstrated here may be important for the biological properties of Fn.
D000911:Antibodies, Monoclonal / Q000276:immunology; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins; D006801:Humans; D011487:Protein Conformation; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D014357:Trypsin