Evidence that the two amino termini of plasma fibronectin are in close proximity: a fluorescence energy transfer study

Wolff, Carl; Lai, Ching San

doi:10.1021/bi00409a053

Cited by 14 publications

(20 citation statements)

References 20 publications

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“…These extended structures also appear difficult to reconcile with the observation that the dramatic change in PFn hydrodynamic behavior at pH 2 or 1 1 is accompanied by only a subtle change in its secondary structure (Alexander et al, 1979;Osterlund, 1988). This model also appears inconsistent with recent fluorescence energy transfer measurements that indicate that the PFn intersubunit distance does not change in the presence of 1 M NaCl (Wolff & Lai, 1988.…”

contrasting

confidence: 90%

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

Benecky¹,

Wine²,

Kolvenbach³

et al. 1991

Biochemistry

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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)

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contrasting

confidence: 90%

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

Benecky¹,

Wine²,

Kolvenbach³

et al. 1991

Biochemistry

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“…Our measurements thus suggest that flexible models based on a string of beads [21] can be rejected with confidence. In any case most of them do not fit with recent evidence demonstrating spatially close N termini [28]. Nevertheless flexibility within the molecule resulting in very rapidly relaxing field-induced orientation (i.e.…”

Section: Discussioncontrasting

confidence: 67%

“…This model, in contrast to flexible structures proposed by others , would fit the hydrodynamic data. This new model would allow an equally good access to the various binding sites of fibronectin, it would explain the relative rates of disulphide bond reductions [36, 371, and permit the close proximity of the two N termini under various ionic-strength conditions [28]. This model is attractive because it would also explain why fibronectin shows some characteristics of a globular molecule with hydrodynamic properties that are incompatible with a compact spherical model.…”

Section: Discussionmentioning

confidence: 99%

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

Vuillard

Roux

Miller

1990

European Journal of Biochemistry

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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

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“…Since no experimental techniques were available to directly probe the loss of tertiary/secondary structure of Fn in cell culture, intramolecular FRET was used to gain conformational information [9,10,21–23]. Here, two Fn labeling schemes for FRET were utilized.…”

Section: Introductionmentioning

confidence: 99%

Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

et al. 2007

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Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.

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Evidence that the two amino termini of plasma fibronectin are in close proximity: a fluorescence energy transfer study

Cited by 14 publications

References 20 publications

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

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

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

Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

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