No abstract

Changes in local environment of the free sulfhydryl groups in plasma fibronectin upon adsorption of the protein to polystyrene beads have been examined by electron spin resonance (ESR) spin-label spectroscopy. The two free sulfhydryl groups per subunit of plasma fibronectin were modified chemically with an [15N, 2H]maleimide spin-label. For soluble fibronectin, both free sulfhydryl groups are shown to be in confined environments as evidenced from the labeled protein exhibiting a strongly immobilized ESR spectrum as described previously using [14N, 1H]maleimide spin-labels [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. When the labeled protein was adsorbed to the beads, half of the strongly immobilized component was found to convert into a weakly immobilized component, a result indicating that one of the two labeled sites becomes exposed and exhibit a fast tumbling motion. Experiments conducted using various spin-labeled fibronectin fragments suggest that the newly exposed labeled site is located between the DNA-binding and the cell-binding regions of the molecule. The data obtained indicate that, upon adsorption to polystyrene beads, plasma fibronectin undergoes a conformational change through which the buried free sulfhydryl group near the cell-binding region of the molecule is exposed. This observation may have important implications regarding the expression of cell adhesive properties of the fibronectin molecule.

These data support a role for both oxidative and nitrosative stress in rejection and the immunoregulatory potential of antioxidant therapy after transplantation.

A fluorescence energy transfer technique has been used to study the intramolecular distance between the two amino termini of human plasma fibronectin. The glutamine-3 residue near the amino terminus of each chain was labeled enzymatically with either monodansylcadaverine or monofluoresceinylcadaverine by use of coagulation factor XIIIa. The nonradiative fluorescence energy transfer between the dansyl (donor) and fluorescein (acceptor) pair in the same protein molecule was determined from steady-state fluorescence measurements. On the basis of the transfer efficiency of 78%, the intramolecular distance between two glutamine-3 residues of fibronectin was estimated to be approximately 23 A, suggesting that the two amino termini of plasma fibronectin are in close proximity. High salt, which affects the hydrodynamic properties of the protein, has no effect on the measured distance. The results support the contention that both compact (in low salt) and expanded (in high salt) conformers of fibronectin are relatively spherical in shape.

Human plasma fibronectin has been investigated by electron spin resonance (ESR) spin-label methods in conjunction with circular dichroism (CD) and sedimentation techniques to investigate its structure and flexibility in solution. The buried sulfhydryl groups of fibronectin were modified with a maleimide spin-label [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. Both conventional and saturation transfer ESR spectra give a rotational correlation time of about (2-3) X 10(-8) s for plasma fibronectin, a value that is at least 40 times faster than the rotational correlation time calculated from the minimal molecular dimensions. This argues that plasma fibronectin is not a compact, globular protein and suggests that the regions of ordered structural domains have a relatively high degree of independent mobility. ESR, CD, and sedimentation measurements showed that many structural features of plasma fibronectin remain unchanged when the pH is decreased from 7.4 to 3.0. On the other hand, ESR results indicate an unfolding of the protein molecule either at pH 11 or in 4 M urea solution. Similarly, the sedimentation coefficient decreases from about 13 to 8.4 S when the pH is raised to 10.8. At pH values above 11, the CD spectrum resembles a random coil; however, some ordered structure is retained either at pH 11 or in 4 M urea. It is likely that the sulfhydryl-containing regions of the molecule are more sensitive to urea or alkali than are portions of the molecule stabilized by intrachain disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)