The measurement of the rotational diffusion coefficient of bovine plasma fibronectin by electric birefringence technique

Abstract

Although the conformation of fibronectin has been widely investigated by various techniques, there has not yet been any determination of its rotational diffusion coefficient. We report here this determination by the transient electric birefringence study of solutions of bovine plasma fibronectin at physiological ionic strength.The solutions showed a positive birefringence. A linear relationship was observed between the intensity of the birefringence at equilibrium and the square of the electric field within the range of fields applied (up to 12.5 kV . cm-I). The field-independent decay of the induced birefringence was described by a single exponential with a relaxation time of 0.76 (f 0.08) ps at 23 "C. This establishes fibronectin in solution as a globally rigid structure with a rotational diffusion coefficient, at 20°C, of 202000 s-'.This result allows the first rigorous determination of the low-resolution structure of fibronectin. It is important to notice that the analysis combines only results obtained in physiological conditions on native molecules and follows a strict hydrodynamic interpretation. The conclusion of this work is that a hollow sphere of about 20 nm external diameter can be proposed as a model for the three-dimensional structure of the fibronectin molecule in solution. This new model suggests the fibronectin could have the structure of a carrier protein.Fibronectins are a class of extracellular glycoproteins (molecular mass: 520 kDa) present in plasma and in the extracellular matrix; for review see [l]. Through the interaction with their receptors (review [2]) fibronectins are an important element in the system that anchors the cells to the tissues. They play an important role in many processes such as embryonic development, wound healing, cancer and metastasis. In solution, fibronectin as studied by light [3,4] and X-ray or neutron [5] scattering techniques does not show any substantial selfaggregation and yet fibronectins are present in the extracellular matrix in an insoluble fibrillar form [6, 71. The physiologically active form (i.e. strong cell binding) is the fibrillar form and the transition between those two forms is not well understood but it has been shown that it involves binding of fibronectin to the cellular receptor and subsequent fibronectin polymerisation [8] through its N-terminal [9]. Thus the study of the conformation of the fibronectin molecule is important to help explain this physiological transition which has a fundamental role in fibronectin function. Abnormal self aggregation, in vitro, of plasma fibronectin from metastatic cancer patients has also been reported [lo].In the absence of a crystal structure, attempts to define the three-dimensional organisation of the fibronectin molecules have relied on electron micrography and hydrodynamic studies in solution.Electron micrographs of fibronectin molecules show two basic pictures of the isolated molecules depending on the sample preparation technique: a V-shaped model with two

Controlling the Spatial Organization of Cells and Extracellular Matrix Proteins in Engineered Tissues Using Ultrasound Standing Wave Fields

“…1). Soluble fibronectin molecules that are 20 nm in diameter (Vuillard et al 1990) experience a maximum primary radiation force of ~4.5×10 −8 pN when exposed to an USWF with parameters shown to promote cell banding (Fig. 3 and Table 1).…”

Solubilization of proteins for electrophoretic analyses

“…The simplest one is carboxybetaine, which is zwitterionic, but only above pH 4, and has been used as a protein solubilizer and stabilizer [144]. More promising are the nondetergent sulfobetaines, whose use in biochemistry was recently introduced [145]. These compounds have been shown to improve solubilization in many biochemical processes, including IEF, while exhibiting no gross denaturing properties [ 145,1461.…”

“…More promising are the nondetergent sulfobetaines, whose use in biochemistry was recently introduced [145]. These compounds have been shown to improve solubilization in many biochemical processes, including IEF, while exhibiting no gross denaturing properties [ 145,1461. Note that these compounds interfere weakly with acrylamide polymerization, which can be overcome by a slight increase in the initiator's concentrations.…”

Influence of temperature and viscosity on anthracene rotational diffusion in organic solvents: Molecular dynamics simulations and fluorescence anisotropy study

“…More recently numerical results for arbitrary shapes have also been obtained. [25][26][27][28][29] Another path involves extracting diffusion coefficients from experimental observables, such as fluorescence depolarization, 30,31 light scattering, 32,33 forced Rayleigh scattering, 34 electric birefringence 35 and nuclear magnetic resonance ͑NMR͒ spectral parameters. 5,[36][37][38] Finally, the advent of computer simulations has opened the door to direct simulations of diffusion, which enable both validation of theoretical models and insight into atomic-level phenomena responsible for experimentally observed effects.…”