Ionic-strength- and pH-dependent conformational states of human plasma fibronectin

Abstract

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 biophysical 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 microseconds) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum protein layers on parylene-C and silicon oxide: Effect on cell adhesion

“…Under acidic, alkaline and high ionic strength conditions, Fn has been reported to acquire a more extended form [38]. An AFM study has revealed that on hydrophilic surfaces, such as silica, most of the protein molecules have an elongated structure whereas they acquire a compact structure on hydrophobic methylated surfaces [39].…”

Adsorption-induced fibronectin aggregation and fibrillogenesis

“…Increasing ionic strength triggers Fn transition from compact to extended form [19][20][21]58] with minimal changes in intra-subunit tertiary structure [21]. The Fn domains that would be involved in the compact conformation of the protein [17,59] are also predictably involved in Fn-Fn intermolecular association [44,45].…”

“…1). Its compact form is stabilized by inter-subunit ionic interactions between III 2-3 and III [12][13][14] or I 1-5 domains [17] and Fn extension may be triggered by ionic strength increase [18][19][20][21] or upon adsorption onto hydrophilic surfaces [22,23]. This transition would allow the exposure of Fn self-association domains required for fibril growth but the mechanisms underlying fibril and subsequent network self-assemblies are still under investigation [10,24,25].…”

Fluorescent Inhibitors for the Qualitative and Quantitative Analysis of Lipolytic Enzymes

“…Fluorescent Marker (19,20) Different amounts (0 to 20 nmol) of pyrenebutanoylsuccinimide ester or perylenebutanoylsuccinimide ester were dissolved in tetrahydrofuran and added to a large excess (3 mg, 45 nmol) of BSA (essentially fatty acid-free, prepared from fraction V, Sigma) dissolved in sodium bicarbonate buffer (0.1 M, pH 8.3) followed by stirring at room temperature for 3 h. All samples contained the same volume of organic solvent (60 L tetrahydrofuran in 1 mL buffer). The reaction scheme is outlined in Fig.…”