Abstract
Human plasma fibronectin ia a high molecular weight (530,000), multi-domain, modular glycoprotein, consisting of two nearly identical subunits disulfide-bridged close to their C-terminal ends. Three sites that can be differentially labeled with fluorescent probes are present on each fibronectin subunit, the transglutaminase-sensitive Gln3 residue and the two free sulfhydryl residues, Cysl201 and Cys2196. These sites are located, respectively, in the N-terminal heparin/fibrin-binding domain, between the central DNA and cell-binding domains, and just before the C-terminal fibrin-binding domain. To map the relative spatial arrangement of these domains, steady-state and lifetime fluorescence energy transfer techniques were employed. Our results show that the minimal intramolecular distances between the labeled Gln3-Cysl201 and Gln3-Cys2196 pairs are 5.5(± 0.6) nm and 5.7(±0.7) nm, respectively, as measured by steady-state methods. Lifetime methods gave somewhat higher distances of 8.1(±0.2)nm and 7.6(±0.2) nm, respectively, between these sites. The binding of heparin or subjection to high ionic strength had only a minor effect, while in the presence of 50% (w/v) glycerol, an increase of about 25% in the intramolecular distances between these sites was observed. A similar effect was induced by binding of fibronectin to the surface of Cytodex beads, an event which was previously shown instead to markedly increase the intersubunit distances between the Gln3-Gln3 and Cysl201-Cysl201 pairs. The solution structure of fibronectin was further investigated by elastic light-scattering and circular dichroism measurements. By elastic light-scattering, the radius of gyration of fibronectin was found to be 15.3(±0.8) nm in the presence of 30% (w/v) glycerol, in contrast to a value of 8.6(±0.3) nm under physiological conditions. Far and near ultraviolet circular dichroism spectra showed that only minor changes in the secondary structure of fibronectin take place on increasing the glycerol content of the solvent up to 34% (w/v). Our results complement previously available information on the solution structure of fibronectin and on its transition from the native compact conformation to a more expanded form on increasing ionic strength or glycerol content. In either situation, fibronectin seems to retain a basic structural core, in which the N-terminal, the central and the C-terminal regions of the two subunits strongly interact with each other. A major role of hydrophobic forces, in stabilizing the fibronectin conformations under these conditions, is therefore postulated. The transition to the extended forms seen in many electron micrographs can instead be explained by disruption of the proposed structural core upon adsorption to surfaces.
| Original language | English |
|---|---|
| Pages (from-to) | 625-640 |
| Number of pages | 16 |
| Journal | Journal of Molecular Biology |
| Volume | 230 |
| Issue number | 2 |
| Publication status | Published - 1993 |
Fingerprint
Keywords
- Extracellular matrix
- Hydrophobic forces
- Intramolecular distances
- Multi-domain proteins
- Protein conformation
ASJC Scopus subject areas
- Virology
Cite this
Solution structure of human plasma fibronectin under different solvent conditions : Fluorescence energy transfer, circular dichroism and light-scattering studies. / Lai, Ching San; Wolff, Carl E.; Novello, Daniela; Griffone, Luca; Cuniberti, Carla; Molina, Francesco; Rocco, Mattia.
In: Journal of Molecular Biology, Vol. 230, No. 2, 1993, p. 625-640.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Solution structure of human plasma fibronectin under different solvent conditions
T2 - Fluorescence energy transfer, circular dichroism and light-scattering studies
AU - Lai, Ching San
AU - Wolff, Carl E.
AU - Novello, Daniela
AU - Griffone, Luca
AU - Cuniberti, Carla
AU - Molina, Francesco
AU - Rocco, Mattia
PY - 1993
Y1 - 1993
N2 - Human plasma fibronectin ia a high molecular weight (530,000), multi-domain, modular glycoprotein, consisting of two nearly identical subunits disulfide-bridged close to their C-terminal ends. Three sites that can be differentially labeled with fluorescent probes are present on each fibronectin subunit, the transglutaminase-sensitive Gln3 residue and the two free sulfhydryl residues, Cysl201 and Cys2196. These sites are located, respectively, in the N-terminal heparin/fibrin-binding domain, between the central DNA and cell-binding domains, and just before the C-terminal fibrin-binding domain. To map the relative spatial arrangement of these domains, steady-state and lifetime fluorescence energy transfer techniques were employed. Our results show that the minimal intramolecular distances between the labeled Gln3-Cysl201 and Gln3-Cys2196 pairs are 5.5(± 0.6) nm and 5.7(±0.7) nm, respectively, as measured by steady-state methods. Lifetime methods gave somewhat higher distances of 8.1(±0.2)nm and 7.6(±0.2) nm, respectively, between these sites. The binding of heparin or subjection to high ionic strength had only a minor effect, while in the presence of 50% (w/v) glycerol, an increase of about 25% in the intramolecular distances between these sites was observed. A similar effect was induced by binding of fibronectin to the surface of Cytodex beads, an event which was previously shown instead to markedly increase the intersubunit distances between the Gln3-Gln3 and Cysl201-Cysl201 pairs. The solution structure of fibronectin was further investigated by elastic light-scattering and circular dichroism measurements. By elastic light-scattering, the radius of gyration of fibronectin was found to be 15.3(±0.8) nm in the presence of 30% (w/v) glycerol, in contrast to a value of 8.6(±0.3) nm under physiological conditions. Far and near ultraviolet circular dichroism spectra showed that only minor changes in the secondary structure of fibronectin take place on increasing the glycerol content of the solvent up to 34% (w/v). Our results complement previously available information on the solution structure of fibronectin and on its transition from the native compact conformation to a more expanded form on increasing ionic strength or glycerol content. In either situation, fibronectin seems to retain a basic structural core, in which the N-terminal, the central and the C-terminal regions of the two subunits strongly interact with each other. A major role of hydrophobic forces, in stabilizing the fibronectin conformations under these conditions, is therefore postulated. The transition to the extended forms seen in many electron micrographs can instead be explained by disruption of the proposed structural core upon adsorption to surfaces.
AB - Human plasma fibronectin ia a high molecular weight (530,000), multi-domain, modular glycoprotein, consisting of two nearly identical subunits disulfide-bridged close to their C-terminal ends. Three sites that can be differentially labeled with fluorescent probes are present on each fibronectin subunit, the transglutaminase-sensitive Gln3 residue and the two free sulfhydryl residues, Cysl201 and Cys2196. These sites are located, respectively, in the N-terminal heparin/fibrin-binding domain, between the central DNA and cell-binding domains, and just before the C-terminal fibrin-binding domain. To map the relative spatial arrangement of these domains, steady-state and lifetime fluorescence energy transfer techniques were employed. Our results show that the minimal intramolecular distances between the labeled Gln3-Cysl201 and Gln3-Cys2196 pairs are 5.5(± 0.6) nm and 5.7(±0.7) nm, respectively, as measured by steady-state methods. Lifetime methods gave somewhat higher distances of 8.1(±0.2)nm and 7.6(±0.2) nm, respectively, between these sites. The binding of heparin or subjection to high ionic strength had only a minor effect, while in the presence of 50% (w/v) glycerol, an increase of about 25% in the intramolecular distances between these sites was observed. A similar effect was induced by binding of fibronectin to the surface of Cytodex beads, an event which was previously shown instead to markedly increase the intersubunit distances between the Gln3-Gln3 and Cysl201-Cysl201 pairs. The solution structure of fibronectin was further investigated by elastic light-scattering and circular dichroism measurements. By elastic light-scattering, the radius of gyration of fibronectin was found to be 15.3(±0.8) nm in the presence of 30% (w/v) glycerol, in contrast to a value of 8.6(±0.3) nm under physiological conditions. Far and near ultraviolet circular dichroism spectra showed that only minor changes in the secondary structure of fibronectin take place on increasing the glycerol content of the solvent up to 34% (w/v). Our results complement previously available information on the solution structure of fibronectin and on its transition from the native compact conformation to a more expanded form on increasing ionic strength or glycerol content. In either situation, fibronectin seems to retain a basic structural core, in which the N-terminal, the central and the C-terminal regions of the two subunits strongly interact with each other. A major role of hydrophobic forces, in stabilizing the fibronectin conformations under these conditions, is therefore postulated. The transition to the extended forms seen in many electron micrographs can instead be explained by disruption of the proposed structural core upon adsorption to surfaces.
KW - Extracellular matrix
KW - Hydrophobic forces
KW - Intramolecular distances
KW - Multi-domain proteins
KW - Protein conformation
UR - http://www.scopus.com/inward/record.url?scp=0027217341&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027217341&partnerID=8YFLogxK
M3 - Article
C2 - 8464068
AN - SCOPUS:0027217341
VL - 230
SP - 625
EP - 640
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -