Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors

Michaela Kreiner, Chandramouli R. Chillakuri, Patricia Pereira, Michael Fairhead, Zhaohui Li, H.J. Mardon, Stephen A. Holt, Christopher F. van der Walle

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Integrin α5β1 binds the human 9th-10th type III fibronectin domain pair (FIII9-10) to mediate cell attachment and spreading. FIII9-10 mutants with increased conformational stability (FIII9010) or highly restricted interdomain mobility (FIII9010-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9-10 was 14% compared to 31% for FIII9010 and 100% for FIII9010-CC. For FIII9010-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9-10 and only a transition to bilayer was observed for FIII9010. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9010-CC were fitted with an integrin layer thickness of 130 A ° . This indicates a movement of the integrin α5β1 headpiece away from its position in the compact 'bent' conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.
Original languageEnglish
Pages (from-to)3954-3962
Number of pages8
JournalSoft Matter
Volume5
DOIs
Publication statusPublished - 2009

Fingerprint

Fibronectins
Integrins
cells
proteins
reflectance
neutrons
Conformations
Neutrons
attachment
isotherms
surfactants
electrostatics
Mutant Proteins
ligands
Surface-Active Agents
Isotherms
Electrostatics
Assays
Ligands
Proteins

Keywords

  • orientation
  • adsorbed fibronectin cells
  • binding domains
  • bound integrin α5β1 receptors
  • pharmacology
  • soft matter

Cite this

Kreiner, M., Chillakuri, C. R., Pereira, P., Fairhead, M., Li, Z., Mardon, H. J., ... van der Walle, C. F. (2009). Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors. Soft Matter, 5, 3954-3962. https://doi.org/10.1039/b908706k
Kreiner, Michaela ; Chillakuri, Chandramouli R. ; Pereira, Patricia ; Fairhead, Michael ; Li, Zhaohui ; Mardon, H.J. ; Holt, Stephen A. ; van der Walle, Christopher F. / Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors. In: Soft Matter. 2009 ; Vol. 5. pp. 3954-3962.
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abstract = "Integrin α5β1 binds the human 9th-10th type III fibronectin domain pair (FIII9-10) to mediate cell attachment and spreading. FIII9-10 mutants with increased conformational stability (FIII9010) or highly restricted interdomain mobility (FIII9010-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9-10 was 14{\%} compared to 31{\%} for FIII9010 and 100{\%} for FIII9010-CC. For FIII9010-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9-10 and only a transition to bilayer was observed for FIII9010. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9010-CC were fitted with an integrin layer thickness of 130 A ° . This indicates a movement of the integrin α5β1 headpiece away from its position in the compact 'bent' conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.",
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Kreiner, M, Chillakuri, CR, Pereira, P, Fairhead, M, Li, Z, Mardon, HJ, Holt, SA & van der Walle, CF 2009, 'Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors', Soft Matter, vol. 5, pp. 3954-3962. https://doi.org/10.1039/b908706k

Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors. / Kreiner, Michaela; Chillakuri, Chandramouli R.; Pereira, Patricia; Fairhead, Michael; Li, Zhaohui; Mardon, H.J.; Holt, Stephen A.; van der Walle, Christopher F.

In: Soft Matter, Vol. 5, 2009, p. 3954-3962.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors

AU - Kreiner, Michaela

AU - Chillakuri, Chandramouli R.

AU - Pereira, Patricia

AU - Fairhead, Michael

AU - Li, Zhaohui

AU - Mardon, H.J.

AU - Holt, Stephen A.

AU - van der Walle, Christopher F.

PY - 2009

Y1 - 2009

N2 - Integrin α5β1 binds the human 9th-10th type III fibronectin domain pair (FIII9-10) to mediate cell attachment and spreading. FIII9-10 mutants with increased conformational stability (FIII9010) or highly restricted interdomain mobility (FIII9010-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9-10 was 14% compared to 31% for FIII9010 and 100% for FIII9010-CC. For FIII9010-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9-10 and only a transition to bilayer was observed for FIII9010. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9010-CC were fitted with an integrin layer thickness of 130 A ° . This indicates a movement of the integrin α5β1 headpiece away from its position in the compact 'bent' conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.

AB - Integrin α5β1 binds the human 9th-10th type III fibronectin domain pair (FIII9-10) to mediate cell attachment and spreading. FIII9-10 mutants with increased conformational stability (FIII9010) or highly restricted interdomain mobility (FIII9010-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9-10 was 14% compared to 31% for FIII9010 and 100% for FIII9010-CC. For FIII9010-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9-10 and only a transition to bilayer was observed for FIII9010. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9010-CC were fitted with an integrin layer thickness of 130 A ° . This indicates a movement of the integrin α5β1 headpiece away from its position in the compact 'bent' conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.

KW - orientation

KW - adsorbed fibronectin cells

KW - binding domains

KW - bound integrin α5β1 receptors

KW - pharmacology

KW - soft matter

UR - http://dx.doi.org/10.1039/b908706k

U2 - 10.1039/b908706k

DO - 10.1039/b908706k

M3 - Article

VL - 5

SP - 3954

EP - 3962

JO - Soft Matter

JF - Soft Matter

SN - 1744-683X

ER -