IONIC-STRENGTH-DEPENDENT AND PH-DEPENDENT CONFORMATIONAL STATES OF HUMAN PLASMA FIBRONECTIN

In order to provide a more detailed understanding of human plasma fibronectin (PFn) solution structure, we examined the effects of pH and ionic strength (mu) variation on the sedimentation velocities (s20,w), fluorescence polarization-derived mean harmonic rotational relaxation times (rho-H), far-ultraviolet (UV) circular dichroism (CD), and intrinsic tryptophan fluorescence of dimeric PFn and the monomeric 190/170-kDa PFn fragment. By comparing the biophysicial properties of PFn with those of the 190/170-kDa PFn fragment, we could assess the relative importance of intrasubunit and intersubunit electrostatic forces in the stabilization of PFn structure. The rho-H derived from isothermal polarization measurements on 1-pyrenebutyrate conjugated PFn decreased markedly (4.5 --> 1.05-1.23-mu-s) when mu was increased from 0.2 to 1.2 or when the pH was adjusted from 7.4 to 2.0 or 11.0. We also noted a significant decrease in the PFn s20,W (13 --> 8.5-9.6S) under these same solvent conditions. In contrast, the rho-H and s20,W of the monomeric 190/170-kDa PFn fragment were relatively insensitive to changes in mu or pH. Computer simulations of the observed pH-dependent changes in the far-UV CD of PFn and the 190/170-kDa PFn fragment revealed only minor differences in protein secondary structure. We also observed only small bathochromic shifts (1-3 nm) in the emission maxima of PFn and 190/170-kDa PFn fragment tryptophan fluorescence under acidic or high mu conditions. These results suggest that minimal changes in PFn tertiary (i.e., intrasubunit) structure occur at pH 2, 11, or at mu = 1.2. We therefore conclude that the driving force behind the observed pH- and mu-induced conformational changes in PFn is disruption of intersubunit electrostatic contacts. Our data suggest that the "unfolded" PFn conformation occurring at pH 2, 11, or mu = 1.2 can be approximated by a structure consisting of two independently rotating disk-shaped subunits, each having a diameter and thickness of 20 and 2.3 nm, respectively.