Structural changes of the cell adhesion protein, fibronectin,in cell culture and on surfaces measured using
fluorescence resonance energy transfer
Loren Baugh
Final ExamSeptember 4, 2003
University of WashingtonDepartment of Bioengineering
Advisor: Viola Vogel Supervisory Committee: Thomas A. Horbett
Patrick S. StaytonFrancois Baneyx
Graduate Student Representative: Mohamed A. El-Sharkawi
Background – The Problem
Proteins in crystals or in solution
Lack of techniques to measure protein structure in physiological environments Lack of knowledge of how proteins work
Background – The Problem
Mechanically-regulated proteins
Mechanical force Biological response
Macroscopic level – Tissue maintenance, diseaseCellular level – Changes in gene expressionMolecular level – How is mechanical force converted to a biochemical signal?
On Off
Protein
Cell
Proteins in crystals or in solution
Lack of techniques to measure protein structure in physiological environments Lack of knowledge of how proteins work
Background – The Problem
Affect the performance of surgical implants, tissue culture substrates, biosensors
How do surfaces control protein activity?
Protein-biomaterial interactions
AdsorptionOn Off
Cell
Protein
Surface A Surface B
Proteins in crystals or in solution
Lack of techniques to measure protein structure in physiological environments Lack of knowledge of how proteins work
Background – Fibronectin
Polymeric fiber, cell adhesive
Cell adhesion, migration, proliferationDevelopment, wound healing
Unregulated fibronectin Fibrotic diseasesLack of fibronectin Cancer metastasis
Cell attachment and growth on biomaterials
Primary biological function: Mediates cell attachment to surrounding environment
Soluble, inactiveBlood, body fluids
Fibronectin
Fibronectin matrix
450 kDa
Single-molecule elasticityModule unfolding / refolding
Background – Fibronectin
Cell
How do structural changes affect the presentation of binding sites?
Unknowns: Structure of Fn in fibers, mechanisms of polymerization, elasticity
Binding sites buried
Binding sites exposed
Fibronectin fibers and single molecules
are elastic
Fibronectin
Overview
Fluorescence resonanceenergy transfer (FRET)
OVERALL GOAL: Study structural changes of fibronectin that control its activity
(1) Calibrate and test energy transfer in solution
(2) Apply to study changes in protein structure in cell culture and on solid surfaces
Insight into how proteins are regulated by mechanical forces and by interactions with biomaterials
Outline
I. Calibration and testing of FRET in solution
II. FRET to measure changes in fibronectin structure in cell culture
III. FRET to measure changes in fibronectin structure on surfaces
Background – Fluorescence resonance energy transferWhat is FRET?
D = DonorA = Acceptor
Normal fluorescence
D = DonorA = Acceptor
Background – Fluorescence resonance energy transferWhat is FRET?
FRET
Dipole-dipole interaction~ Through-space NOESY in NMR~ Two antennae
FRET is sensitive to nanometer-scale changes in donor-acceptor distance
BackgroundWhy is FRET useful?
“Spectroscopic ruler”
BackgroundWhy is FRET useful?
Sensitive range
Typical Ro = 2-7 nmShown: Ro = 6 nm
dos Remedios, Moens.J Struct Biol (1995)115(2): 175-185
Structural changes undetected
Background – FRET between a single donor-acceptor pairis not sensitive to full range of structural changes of a LARGE protein
(Ro = 2 - 7 nm)
Background – FRET between a single donor-acceptor pairis not sensitive to full range of structural changes of a LARGE protein
Structural changes undetected
50:50 mixturedonors and acceptors
buffer, pH 7, RT, 4 h
25%
25%
25%
25%
~ 100% efficient 2 D, 2 A per Fn
MethodsLabel fibronectin so FRET is sensitive to its structural changes
Labeling Scheme 1 : Donors + acceptors on free cysteines (D/Acys)
Fibronectin
~ 160 nm
SH SH
SH SH
buffer, pH 7, RT, 4 h
25%
25%
25%
25%
> 10 nm~ 100% efficient 2 D, 2 A per Fn
50:50 mixturedonors and acceptors
Outside range of FRET
MethodsLabel fibronectin so FRET is sensitive to its structural changes
Labeling Scheme 1 : Donors + acceptors on free cysteines (D/Acys)
~ 160 nm
SH SH
SH SH
Step 1: Cysteine-reactive donors, pH 7, 4 h
4 D, 7 A per FnStep 2: Amine-reactive acceptors, pH 8.3, 4 h
Increase number of donor-acceptor pairs per protein
Increase sensitivity of FRET
MethodsLabel fibronectin so FRET is sensitive to its structural changes
Labeling Scheme 2 : Donors on free cysteines, acceptors on random amines (Dcys Aamine)
Fibronectin
~ 160 nm
SH SH
SH SH
Step 1: Cysteine-reactive donors, pH 7, 4 h
4 D, 7 A per FnStep 2: Amine-reactive acceptors, pH 8.3, 4 hFibronectin
~ 160 nm
Oregon Green 488 Tetramethylrhodamine
Donor Acceptor
1.1 nm 1.5 nm
Largest fluorophore and Fndrawn to scale
MethodsLabel fibronectin so FRET is sensitive to its structural changes
Labeling Scheme 2 : Donors on free cysteines, acceptors on random amines (Dcys Aamine)
SH SH
SH SH
Step 1: Cysteine-reactive donors, pH 7, 4 h
4 D, 7 A per FnStep 2: Amine-reactive acceptors, pH 8.3, 4 h
Alexa Fluor 488 Alexa Fluor 594
Donor Acceptor
1.0 nm 1.8 nm
Largest fluorophore and Fndrawn to scale
MethodsLabel fibronectin so FRET is sensitive to its structural changes
Labeling Scheme 2 : Donors on free cysteines, acceptors on random amines (Dcys Aamine)
SH SH
SH SHFibronectin
~ 160 nm
Secondary structure (-sheet)
Test FRET vs. known structural changes
Denaturant
[Guanidine-HCl] (M)0 1 2 8
Fn
Circular dichroism spectroscopy
Part I : Calibration and testing of FRET - Results
[Guanidine-HCl] (M)1 2 80
Part I : Calibration and testing of FRET - Results
Test FRET vs. known structural changes
Labeling scheme 2: Dcys Aamine
1 2 80
Part I : Calibration and testing of FRET - Results
Test FRET vs. known structural changes
[Guanidine-HCl] (M)
Labeling scheme 2: Dcys Aamine
Part I : Calibration and testing of FRET - Results
Test FRET vs. known structural changes
FRET
Secondary structure (-sheet)
D/Acys
1 2 80
Dcys Aamine
FRET is sensitiveto a larger rangeof structural changes
[Guanidine-HCl] (M)
Dcys Aamine
Part I : Calibration and testing of FRET - Results
Test FRET vs. known structural changes
NaCl
Dithiothreitol
Thermolysin
1 M, RT, 1 h
0.5 M, RT, 1 h
1 mg/mL, 70° C, 4 h
Changes in FRET are consistent with known changes in fibronectin structure
D/AcysDcys Aamine
Part II : FRET to measure fibronectin structure in cell cultureMethods
Rinse, Fix cells,
Mount samples
0.5 h 1-24 h
37° C 37° C
FRET microscopy / spectroscopy
+
Plate cells
NIH 3T3 fibroblasts
Add labeledprotein
Excessunlabeled protein
to preventinter-molecularenergy transfer
10 g/mL Fn**90 g/mL Fn
Plate cells
NIH 3T3 fibroblasts
0.5 h 1-24 h
37° C 37° C
Add labeledprotein
Excessunlabeled protein
to preventinter-molecularenergy transfer
10 g/mL Fn**90 g/mL Fn
FRET microscopy / spectroscopy
FRET microscopy / spectroscopy
Disrupt celltension
Cytochalasin D, 10 M
1 h
37° C
Rinse,Fix,
Mount
Part II : FRET to measure fibronectin structure in cell cultureMethods
+
Slit, Mirror Slit, Grating
Wavelength
Pos
ition
Window, Mirror
~ 1 m
D A
Color Structure
Excite donors,collect donor (green) and acceptor (red)emission
Methods – FRET microscopy and spectroscopy
Imaging Spectroscopy
Results – FRET microscopy
Red High IA / ID High FRET Compact
Green Low IA / ID Low FRET Extended
Excite donors, collect donor and acceptor emission
Fibronectin is more extended in fibers than on the cell surface
D
A
I
Cell surface (red)
Fibers (green – range of values)
10 m
Color ofemissionin image
Proteinstructure
Baneyx, Baugh, Vogel. PNAS (2001) 98(25): 14464-14468.
D
A
I
Cell surface (red)
Fibers (green, range of values)
10 m
Results – FRET spectroscopy
Fibronectin is compact on the cell surface
Fibronectin exists in a range of extended conformations in fibers
Spectra in black: Labeled Fn in 0, 1, 8 M guanidine-HCl solutions
Fibronectin is converted by cells into an extended structure in early stages of polymerization
Red Compact
Green Extended
Structure changes during polymerization
Populations ofprotein
Results – Time-lapse FRET imaging
Extension of Fnby cell receptors exposes Fn-Fn binding sites
Schwarzbauer, Sechler.Curr Opin Cell Biol (1999)11(5): 622-6270.
Fibronectin Polymerization
Fibers are pulled out of stationary fibronectin clusters by cell integrins, driven by the actin cytoskeleton
Pankov et al. J Cell Biol(2000) 148(5): 1075-1090.
Fibronectin Polymerization
Role of mechanical force in polymerization
Cytochalasin D
10 M
1 h37° C
Disrupt cell tension
I Fibers Range of FRET
Measure FRET from 1 m segments along fibers of treated and untreated cells
FRET to study the structure of fibronectin in fibers
Test effect of cell tension on conformation of fibronectin in fibers
Range of conformations
IAID
Distance along fiber (m) • FRET was uniform along some fibers, varied along others
• Mean FRET level in fibers was low fibronectin extended
Cytochalasin D
Baneyx, Baugh, Vogel. PNAS (2002) 99(8): 5139-5143.
FRET to study the structure of fibronectin in fibers
Conformation of fibronectin in fibers in untreated cells
Untreated
10 m
IAID
Distance along fiber (m)
Buffer
8 M Guanidine
IAID
Distance along fiber (m)
IAID
Distance along fiber (m)
• Treatment with cytochalasin D caused an increase in FRET in fibers
Release of tension allowed fibronectin in fibers to refold
Untreated Cytochalasin D
C
Buffer
8 MGuanidine
Cyto-chalasin D
10 m
Untreated
Baneyx, Baugh, Vogel. PNAS (2002) 99(8): 5139-5143.
FRET to study the structure of fibronectin in fibers
Conformation of fibronectin in fibers in cells treated with cytochalasin D
Intermediate FRET
Low FRET
Fibronectin fiber elasticityModule unfolding / refolding
Single-molecule AFM force-spectroscopy
Fn-Fn sliding
Fibronectin un-bending / re-bending
Module unfolding / refolding
Intermediate FRET
Low FRET
High FRET
Intermediate FRET
Intermediate FRET
Intermediate FRET
Fibronectin fiber elasticityModels of elasticity
Flattening of the RGD loop and loss of cell integrin binding activity
Fibronectin as a force-regulated adhesive switch
Appliedforce
Fixed
Steeredmoleculardynamicssimulations
Functional consequences of module unfolding / refolding
Krammer et al.PNAS (1999) 96(4): 1351-1356.
FnIII-10FnIII-9
Appliedforce
Fixed
Regulation of proteins by mechanical force
• 2% of all animal proteins share fibronectin’s force-sensitive FnIII modules
• Many adhesion proteins share the RGD loop
Module unfolding and refolding – Common mechanism of elasticity and force-regulated protein function?
FnIII-10FnIII-9
Proteins adsorbed to biomaterialsOverall picture of protein structure on surfaces is limited
Adsorption
Antibody binding
ElectronMicroscopy
Fibronectin
Circular dichroism spectroscopyDifferential scanning calorimetry Tryptophan fluorescenceAtomic force microscopy
Other techniques
Artifacts:Protein sprayed
from glycerolsolution
FRET spectroscopy
1 h, RT
Add labeledand unlabeled
fibronectin
Rinse by serial dilution Measure FRET, Total fluorescence
intensity
0.1 – 25 g/mL (total)
FRET to measure fibronectin structure adsorbed to surfacesMethods
SurfacePreparation
Adsorption
Glasscoverslips Sulfuric acid
No-ChromixFluorosilanedeposition
SiO- CF3(CF2)5(CH2)2Si
Eliminate inter-molecular energy transfer
adv = 5adv = 110
FRET to measure fibronectin structure adsorbed to surfacesResults
(Density)Fn on fluorosilane > (Density)Fn on glass
Amount offibronectinadsorbed
Undesired energy transfer between adjacent adsorbed proteins
Labeled Fn + unlabeled Fn = 25 g/mL
Labeled fibronectin
only
Results – FRET spectroscopy
Labeled Fn + unlabeled Fn = 25 g/mL
Labeled fibronectin
only
Inter-molecular energytransfer eliminated
Lower FRET on glass
Fibronectin is more extended on glass
Results – FRET spectroscopy
Adsorption
Glass Fluorosilane
Strong hydrophobic interactions trap Fn in a compact state
Fibronectin-surface interactions
SiO- CF3(CF2)5(CH2)2Si
FRET results Fibronectin is more extended on glass than fluorosilane
Hydrophilic, negatively charged Hydrophobic, neutral
Charge-charge interactions disrupt Fn’s compact state
Previous studies Antibody binding Secondary structure – circular dichroism, calorimetry
How do surface interactions affect fibronectin’s cell adhesion activity?
Adsorb labeledfibronectin
1, 25 g/mL
1 hRT
Rinse Plate cells45 min37C
Remove unbound cells
NIH 3T3 fibroblasts
Serum-free medium, 10 mg/mL albumin
(block non-specific adhesion)
Count
Cell attachment – MethodsCompare Fn’s cell adhesion activity on glass and fluorosilane
Glass1 g/mL, Labeled Fn
Fluorosilane1 g/mL, Labeled Fn
Imageof cells
Cell attachment – Results
Why was cell attachment lower on fluorosilanedespite a greater density of adsorbed Fn than on glass?
Why the concentration-dependence?
AdsorptionCell
Glass Fluorosilane
Fibronectin’s conformation on glass was favorable for cell attachment
Above
Above Above
Below
Density of exposed sites (above/below threshold for attachment)
• Minimum density of exposed integrin binding sites required for cell attachmentMassia, Hubbell. J Cell Biol (1991) 114(5):1089-100.
• Fibronectin more extended on glass Greater avg exposure of RGD sites
• (Density)Fn on fluorosilane > (Density)Fn on glass
Summary of Results
IAID
Cell surfaces
Synthetic materials
Extracellular matrix fibers
Solution
PBS NaCl 1 MGdn
DTT 8 MGdn
4 MGdn
Future Directions
• FRET microscopy and spectroscopy applied to other proteins
Spatial resolution of different protein conformations
• FRET combined with site-directed mutagenesis
Novel labeling sites in strategic locations
Conclusions
Native structures Structures on biomaterials
Force - regulated Unregulated
Compact,Inactive
Extended,Adhesive
IntermediateConformations
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