Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.

In vitro denaturation-renaturation of fibronectin. Formation of multimers disulfide-linked and shuffling of intramolecular disulfide bonds.

It is well established that fibronectin into extracellular matrix undergoes repeated tensions applied by cells, resulting into dramatic structural changes which reflect its elastic properties. However, there is currently no study reporting with precision the consequences of this elasticity on fibronectin structure and conformation. In the present work, we investigated fibronectin structural and conformational reorganization in vitro through a denaturation-renaturation approach. The similarities and differences between "refolded fibronectin" and "native fibronectin" were investigated using various spectroscopic methods, hydrodynamic characterization, molecular imaging and biochemical characterization. In the refolded form, secondary structure elements as well as local tyrosine and tryptophan environment are identical compared to the native form. Interestingly, some differences in global tertiary structure organization and molecular conformation were observed. These differences are due to the reactivity of the two free cysteines, which are buried in the native state but become accessible during the unfolding process. First, oxidation of these residues leading to the formation of intermolecular disulfide bonds results in formation of stabilized multimer. Second, some illegitimate intramolecular disulfide bonds are formed. The presence of iodoacetamide, the sulfhydryl alkylating agent, during the unfolding-refolding process prevents all these events. This study clearly demonstrates that, under near physiological conditions, competitive renaturation pathways occur, involving free cysteines in either multimer formation or intermolecular shuffling of disulfide bonds. These findings might have important implications for future studies and be helpful to develop a deeper understanding of fibronectin morphology.

Urea-induced sequential unfolding of fibronectin: a fluorescence spectroscopy and circular dichroism study.

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 <= => I(1) <= =>I(2) <= => U. Fitting this model to experimental values, we have determined the free energy change for the different steps. We found that the N <= => I(1) 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 I(1) <= => I(2) 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, I(2) <= => 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.