Human plasma fibronectin structure probed by steady-state fluorescence polarization: evidence for a rigid oblate structure

Abstract

In order to more clearly define the structure of human plasma fibronectin (PFn) under physiologic buffer conditions, we determined the mean harmonic rotational relaxation times (rho H) of PFn and the thrombin-derived 190/170-kDa PFn fragment using steady-state fluorescence polarization. These measurements utilized the long lifetime emission (tau = 1.2 X 10(-7) S) exhibited by 1-pyrenebutyrate, which had been covalently attached to amino groups at random sites on the PFn subunit. Our data analysis assumed that two independent processes depolarize the fluorescence exhibited by the dansylcadaverine and 1-pyrenebutyrate conjugates of PFn: (A) rapid (rho H less than 10(-9) S) "thermally-activated" localized rotational motion of the protein side chains bearing the fluorescent probe [Weber, G. (1952) Biochem. J. 51, 145-154] and (B) slow (rho H approximately 10(-6) S) temperature-independent global rotational motion of the whole PFn molecule. Since only the rho H associated with the latter process is a true hydrodynamic parameter (i.e., sensitive to size and/or shape of the PFn molecule), we utilized isothermal polarization measurements to discriminate against the interfering signal arising from "thermally activated" probe rotation. The rho H (4.4 +/- 0.9 microseconds) derived from an experiment in which pyrene-PFn fluorescence polarization was monitored as a function of sucrose concentration at constant temperature is 7 (+/- 1.4) times longer than that predicted for an equivalent hydrated sphere. We propose that "thermally activated" probe rotation gives rise to the nearly 100-fold shorter PFn rho H values previously reported in the literature. Consequently, our data exclude all previous models which invoke segmental flexibility of the PFn peptide backbone. The simplest hydrodynamic model supported by our fluorescence data is an oblate ellipsoid with an axial ratio of 15:1. All prolate models can be unambiguously excluded by this result. We estimate that the disk-shaped PFn molecule has a diameter and thickness of 30 and 2 nm, respectively. Electron microscopy of negatively stained PFn specimens on carbon also showed PFn to have a compact rounded structure. The much faster rotational relaxation rate of the pyrene-190/170-kDa PFn fragment (rho H = 0.92 +/- 0.11 microseconds) compared to pyrene-PFn indicated that this monomeric PFn fragment, like native PFn, had an oblate shape under physiologic buffer conditions.

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

“…Structures observed during electron-microscopic examination of PFn specimens that had been deposited on carbon films and visualized by negative staining (Benecky et al, 1990) or STEM (Tooney et al, 1983) appear consistent with this model.…”

“…Therefore, this fragment lacks the 30-kDa amino-terminal domain. We have previously shown that this large monomeric PFn fragment retains all of the gelatin-and heparin-binding activity exhibited by the native dimeric molecule (Benecky et al, 1988(Benecky et al, , 1990.…”

“…The 1-pyrenebutyrate conjugates of PFn and the 190/170-kDa PFn fragment were prepared by the use of the amine-reactive reagent succinimidyl-1-pyrenebutyrate (Benecky et al, 1990). Gelatin-Sepharose 4B-CL affinity chromatography was employed to separate PB-PFn and PB-190/170 kDa from unreacted succinimidyl-1-pyrenebutyrate (Benecky et al, 1990). We have previously shown that the labeling reaction with 1-pyrenebutyrate is not confined to any specific region on the PFn subunit (Benecky et al, 1990).…”

“…Gelatin-Sepharose 4B-CL affinity chromatography was employed to separate PB-PFn and PB-190/170 kDa from unreacted succinimidyl-1-pyrenebutyrate (Benecky et al, 1990). We have previously shown that the labeling reaction with 1-pyrenebutyrate is not confined to any specific region on the PFn subunit (Benecky et al, 1990). The number of dye molecules bound per protein molecule was determined by absorption spectroscopy using a molar extinction coefficient of 4.0 X lo4 M-' cm-' at 346 nm for protein-bound 1-pyrenebutyrate (Knopp & Weber, 1969).…”

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.…”