Urea-Induced Sequential Unfolding of Fibronectin: A Fluorescence Spectroscopy and Circular Dichroism Study

Urea-Induced Sequential Unfolding of Fibronectin: A Fluorescence Spectroscopy and Circular Dichroism Study
Received May 2, 2003
Revised Manuscript Received December 10, 2003
Web Release Date: January 21, 2004
Salima Patel, Alain F. Chaffotte, Fabrice Goubard, and Emmanuel Pauthe*
Biochemistry
ACS Publications
Copyright ? 2005 American Chemical Society
ERRMECE, Universit? de Cergy-Pontoise, 95302 Cergy-Pontoise Cedex, France, LPPI, Universit? de Cergy-Pontoise, 95301 Cergy-Pontoise Cedex, France, and Institut Pasteur, 28 rue du Dr. Roux, Paris, France
Abstract:
Fibronectin (FN) is an extracellular matrix (ECM) protein found soluble in corporal fluids or as an insoluble fibrillar component incorporated in the ECM. This phenomenon implicates structural changes that expose FN binding sites and activate the protein to promote intermolecular interactions with other FN. We have investigated, using fluorescence and circular dichroism spectroscopy, the unfolding process of human fibronectin induced by urea in different ionic strength conditions. At any ionic strength, the equilibrium unfolding data are well described by a four-state equilibrium model N I1 I2 U.Fitting this model to experimental values, we have determined the free energy change for the different steps. We found that the N I1 transition corresponds to a free energy of 10.5 ? 0.4 kcal/mol. Comparable values of free energy change are generally associated with a partial unfolding of the type III domain. For the I1 I2 transition, the free energy change is 7.6 ? 0.4 kcal/mol at low ionic strength but is twice as low at high ionic strength. This result is consistent with observations indicating that the complete unfolding of the type III domain from partially unfolded forms necessitates about 5 kcal/mol. The third step, I2 U, which leads to the complete unfolding of fibronectin, corresponds to a free energy change of 14.4 ? 0.9 kcal/mol at low ionic strength whereas this energy is again twice as low under high ionic strength conditions. This hierarchical unfolding of fibronectin, as well as the stability of the different intermediates controlled by ionic strength demonstrated here, could be important for the understanding of activation of the matrix assembly.
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