Force Microscopy Studies of Fibronectin Adsorption and Subsequent Cellular Adhesion to Substrates With Well-Defined Surface Chemistries

Pamela Meadows, Gilbert Walker Abstract Molecular force spectroscopy was used to study the mechanical behavior of plasma fibronectin (FN) on mica, gold, poly(ethylene) glycol (PEG), and -CH3, -OH, and -COOH terminated alkanethiol self-assembled monolayers. Proteins were examined at two concentrations, one resulting in a saturated surface with multiple intermolecular interactions referred to as the aggregate state and another resulting in a semiaggregate state where the proteins were neither completely isolated nor completely aggregated. Modeling of the force-extension data using two different theories resulted in similar trends for the fitted thermodynamic parameters from which insight into the protein’s binding state could be obtained. Aggregated proteins adsorbed on hydrophobic surfaces adopted more rigid conformations apparently as a result of increased surface denaturation and tighter binding while looser conformations were observed on more hydrophilic surfaces. Introduction

Fibronectin (FN) is a large multidomain protein that is found on cell surfaces, in plasma, and other body fluids as well as being a major constituent of the extracellular matrix (ECM). Of particular interest is the elasticity of this protein. It is believed to be crucial for the formation of fibronectin fibrils in the ECM which in turn can increase cell and molecular adhesion to the surface. In this study, atomic force microscopy is being used to study fibronectin’s elasticity to obtain new insights into its structural properties and how these properties are influenced by the surface. By understanding how the surface influences the protein’s conformation, new biomaterials can then be designed. Background to Single Molecule Force Stretching Mechanics: Forcing Homopolymer Conformational Change 0.05 1

0 0 2 4 Force, nN 3 -0.05 Tip-Sample Separation

force [nN] 2 (nm) -0.1 4 Above is a representation of an ideal force plot where a -0.15 polymer on the surface 1 3 interacts with an AFM tip. To -0.2 the right is a schematic 10 20 30 40 50 60 70 Tip-sample separation [nm] showing the different stages of Worm-like Chain kT 1 1 this process and the fitting that F = [ - + R ] Model is used. q 4(1 - R ) 4 Protein Stretching and Unfolding via AFM Step 1 Step 2 Step 3

Tip Tip Tip

Surface Surface Surface

Step 4 Step 5 Step 6

Repeat step 3

Surface Surface Surface Force Plot of a Multidomain Protein Unfolding

1 Extend 0 2 4 5 Retract

Force, nN Force, 3 3' 6

Tip-Sample Separation, nm

3 and 3' represent domain unfolding while 6 represents protein-tip rupture Characteristics of Fibronectin

Fibrin Heparin DNA DNA Collagen Cells Bacteria Heparin Heparin Heparin Fibrin SH SH P I I I I I I II II I I I III III III III III III III III III III III III III III III III III I I I NH2 C SS

Domains Type Type Type z Adhesion promoting protein I II III z 2446 amino acids # in FN 12 2 17 z 140 nm in length and 2nm in diameter Avg # of 45 60 90 z Dimer linked via disulfide bond res. (monomer shown above) Max. 16nm 22nm 33nm length What unfolding events do we see?

Fibronectin, Ed, D.F. Mosher (San Diego, Academic Press 1989) Type III domains lack disulfide bonds and therefore unfold with our loading rates. Experimental Conditions/Procedure General

1. Prepared a 50μg/mL FN solution in PBS (10mM Na2HPO4, 2.7mM KCl, 138mM NaCl). 2. Monomer prepared by using 35μg/mL FN solution in 150mM NaCl, 1mM EDTA, 1mM

NaN3, 5mM DTT, and 20mM KH2PO4. Solution was then run through a NAP-10 desalting column with 20mM KH2PO4, 150mM NaCl, and 1mM EDTA as the equilibration buffer before AFM measurements were made.

3. Si3N4 tips cleaned in dilute HF with filtered NANO pure water (≤ 18MΩ) for 1.5 hours. Surface Preparation 1. Monolayers were grafted on gold by immersion in 1mM end functionalized alkane thiol in ethanol, overnight, followed by rinsing in pure solvent.

2. Glass substrates cleaned at least 12 hours in a bath of 500g KOH, 500ml H2O, and 4L iso- Propanol. 3. Surfaces placed in approximately 1mL of FN (dimer) solution at 4°C for 18-24 hours and attached to a specimen disk via epoxy. Monomer left on surface for 10 minutes.

Data Collection Samples imaged in fluid (PBS) using a stand-alone molecular force probe (MFP).

Data Analysis All analysis was performed using custom written software in Matlab. Unfolding Isolated Single Proteins: transition length difference less than domain contour length

0.6 50

45 0.4 40 ces

en 35 r

0.2 r )

cu 30 c

0 O 25 f

o 20 Force (nN -0.2 15

6.1 mber

nm u

N 10 -0.4 5

0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 90 Tip-Sample Separation (nm) Length (nm)

The left panel shows a force plot obtained when extending single molecules of FN away from a mica surface in water. The length intervals between successive ruptures was determined by subtracting the difference in tip-sample separations at points prior to the cantilever returning to near zero force, as illustrated by the vertical lines. The right panel gives a histogram of these length intervals the most probable value of 9.5 ± 0.5nm was determined by a Lorentzian fit. Unfolding Protein Domains in Aggregated proteins: Transition length difference matches domain contour length

20 0.8

0.6 15

0.4

10 0.2 Force (nN)

0 5 Number of Occurrences

-0.2

0 0 50 100 150 200 250 300 10 15 20 25 30 35 40 45 50 Tip-Sample Separation (nm) Length (nm)

Conclusion: Aggregation protects domains from unfolding on surface. Force Spectroscopy Reveals Barriers to Unfolding: Bell-Evans Model

‡ ‡ )( −Δ=Δ FxGFG B ⎛ hk ⎞ ‡ ⎜ off ⎟ −=Δ BTkG ln⎜ ⎟ ⎝ BTk ⎠ BTk Slope = After Evans, E. Faraday Disc. 1998, 111, 1-26. xB • Unfolding is an activated process. From the slope and y- intercept of a loading rate • With forced unfolding, inner barriers vs. rupture force plot, xb can be observed. and koff can be determined. Rupture forces: Comparing aggregates and isolated molecules 0.4 0.4 0.4

0.35 A) 0.35 B) 0.35 C)

0.3 0.3 0.3

0.25 0.25 0.25

0.2 0.2 0.2 Force (nN) Force (nN) Force (nN) Force 0.15 0.15 0.15

0.1 0.1 0.1

0.05 0.05 0.05 100 101 10 2 100 101 102 100 10 1 102 Loading Rate (nN/sec) Loading Rate (nN/sec) Loading Rate (nN/sec) •Plot A shows measurements performed on single FN molecules isolated on a mica substrate in water. •Plot B represents FN densely deposited on a glass substrate in PBS. In both A and B, the last rupture in the force plots, which corresponds to rupture of the protein from the tip, is excluded from the analysis. •In Plot C, rupture forces in plots with only one pulling event are analyzed for isolated single FN molecules on mica in water, which therefore includes the protein-tip rupture. •Under the Bell model and its extensions, each linear region corresponds to a barrier crossing process. Changing the Substrate: Surfaces Studied and Observed Force Events Substrate Total % of Force Number of Ruptures Number of Force Plots Used in Analysis of Single Plots Displaying Multi-Rupture Force Rupture Collected Stretching Plots Force Plots Events Mica 6967 15.2 % 2376 306

Gold 6952 17.7 % 2062 377

11-Mercapto- 7471 21.9 % 2828 542 1-undecanol 11-Mercapto- 8012 11.8 % 2059 283 undecanoic acid 1- 9003 8.9 % 1626 280 Hexadecanethiol PEG* 9002 0.7 % 155 23

*Few aggregated proteins were found on this substrate due to its resistance to protein adsorption Using the Hummer Model to Analyze Protein-Surface Rupture

AFM tip • Adhesion bond is loaded by an effective spring that is created by

km* the combined springs of the cantilever and protein. • The effective spring constant, k *, is obtained by fitting a line to Substrate s the steeply sloped region of a force plot. free energy • Bond spring constant is km* and provides the curvature in the free energy surface seen in this figure. ks*

0 Hummer, G.; Szabo, A. Biophys. J., 2003, 85, 5. bond extension, x Hummer-Szabo Model, cont’d

• A molecular free energy surface (Vo(x)) whose potential of mean force is given by = o + s − vt)(xV(x)VV(x,t) • Here, the reaction coordinate, x, is coupled to the AFM’s piezo velocity, v. The free energy is ⎧1 2 ⎫ ⎪ m < β )x(xxk ⎪ ⎪2 ⎪ β o(x)V = ⎨ ⎬

⎪ ≥∞− β )x(x ⎪ ⎩⎪ ⎭⎪ -1 • above km represents the molecular spring constant, β = kBT, and xβ again corresponds to the distance from the free energy minimum to the transition state projected along the direction of applied force. Since it is assumed that the system undergoes Brownian motion on the free energy surface, Kramers theory provides the relationship between the rate of rupture in the absence of pulling (koff(0)) and the system’s properties through the equation below 2 / 23 ‡ )(xk 0 ≈ 2π)();k)(x(k − / 21 exDk Δ− GB β G ‡ =Δ m β off β m m β where 2 • Here D and ΔG‡ represent the diffusion coefficient and the unfolding barrier height, respectively. Rupture force vs. velocity, cont’d / 21 γ+ 2 /)(xk 2 __ ⎡ m β ⎤ off 0 )e(k β m β −= ⎢ kxkF ln2 ⎥ += kkk, sm ⎢ /k)(kvxk / 23 ⎥ ⎣ s β m ⎦

• A fit of the average rupture force ( F ) versus velocity (v) allows the molecular spring constant (km* = kBTkm), the barrier distance (xβ), and the kinetic offrate (koff(0)) in the absence of pulling to be obtained. • ks is an effective spring constant that incorporates the spring constants of both the cantilever and the extended chain; ks* = kBT ks, and kchain and kc represent fibronectin and the cantilever’s spring constant, respectively

⎛ + kkkk chaincmc ⎞ ⎜1+ ⎟ • a factor of ⎝ kk chainm ⎠ was multiplied to v in the above equation, to account for the tip’s velocity Thermodynamic Parameters for the Forced Extension of Semiaggregated Fibronectin Using Force Plots Containing A Single Rupture

Substrate Bell/Evans Model Hummer Model#

‡ * ‡ xβ koff(0) ΔG (0) ks km xβ koff(0) ΔG (0) D (nm) (s-1) (kcal/mol) (nm-2) (N/m) (nm) (s-1) (kcal/mol (nm2/s ) ) Mica 1.38 ± 0.45 0.01 ± 0.04 19.9 ± 2.0 1.4 0.41 0.29 135 2.5 81 0.09 ± 0.01 49 ± 20 15.0 ± 0.2

11-Mercapto- 0.09 ± 0.03 24 ± 20 15.5 ± 0.5 2.7 0.94 0.18 66 2.3 13 1-undecanol 11-Mercapto- 0.14 ± 0.01 13 ± 3 15.7 ± 0.1 5.3 3.1 0.10 46 2.3 3 undecanoic acid 0.02 ± 3E- 51 ± 43 14.9 ± 0.5 3 1- 0.11 ± 0.01 22 ± 7 15.4 ± 0.2 2.6 1.5 0.17 62 3.1 27 Hexadecane thiol Gold 0.08 ± 0.02 11 ± 10 15.9 ± 0.6 5.4 0.87 0.28 0.81 4.8 9

In single rupture force plots, the surface-protein adhesion is probed Fibronectin’s Rigidity (kchain) when Semiaggregated on a Substrate

* Substrate Force Plot Type kchain, (nN/nm)

Mica Multi-rupture 7.9E-3 ± 5.7E-4 Single rupture 6.2E-3 ± 4.7E-4 11-Mercapto-1-undecanol Multi-rupture 1.3E-2 ± 5.0E-4 Single rupture 1.4E-2 ± 1.2E-3 11-Mercaptoundecanoic Multi-rupture 3.1E-2 ± 1.2E-3 acid Single rupture 3.1E-2 ± 1.9E-3 1-Hexadecanethiol Multi-rupture 1.2E-2 ± 4.9E-4 Single rupture 1.2E-2 ± 4.4E-4 Gold Multi-rupture 3.9E-2 ± 1.4E-3 Single rupture 4.2E-2 ± 1.8E-3 Correlations obtained in the aggregate (1, 2, 4) and semiaggregate (3) experiments for force plots containing a single (1-3) or multiple (4) elastic response

1255 55 mica mica y = -3302x + 65 50 50 y = -71.3x + 59.2 •As FN’s rigidity (kchain) -OH -OH 45 45 or interfacial stiffness -COOH -COOH * 40 40 (km ) increases, the length

35 35 -CH3 -CH3 to which the chain can be Length (nm) Length (nm)

30 30 extended via AFM Au Au 25 25 decreases.

20 20 2 4 6 8 10 12 14 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 -3 kchain (nN/nm) x 10 km* (nN/nm) •For these plots, peak 0.12 24 lengths and rupture forces 340.11 y = 1.9x + 0.02 22 y = -808.4x + 25.8 Au mica were obtained by a 0.1 20 Lorentzian or Gaussian fit 0.09 -OH 18 -CH3 0.08 to the data. -COOH 0.07 16 -COOH L (nm) 0.06 Force (nN) -OH 14 •For the last figure, one 0.05 PEG 12 0.04 -CH3 data point, representing 10 Au 0.03 mica PEG, was omitted from 0.02 8 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0 0.005 0.01 0.015 0.02 0.025 0.03 the fit. kchain (nN/nm) kchain (nN/nm) Conclusions

¾ Aggregated proteins adsorbed on hydrophobic surfaces adopted more rigid conformations while looser conformations were observed on more hydrophilic surfaces. ¾ Studies of FN in a semiaggregate state showed heterogeneity in the model’s thermodynamic parameters suggesting that in the early stages of nonspecific adsorption, multiple protein conformations exist, each having bound irreversibly to the substrate. ¾ Diffusion coefficients we obtained are roughly similar to those found in protein spreading experiments on albumin and fibrinogen.*

*Wertz, C. F.; Santore, M. M. Langmuir, 2001, 17, 3006 AFM’s Correlation With Cell Deposition Experiments: Aggregated FN Enhances Cell Seeding

• Fluorescent images (nuclear staining; image size = 2.5 mm2) of

HUVEC cells deposited on –CH3 terminated SAMs with FN in a semiaggregate (Left; Cell count = 365) and aggregate form (Right; Cell Count = 993). FN in an aggregate form promoted cell deposition and proliferation while FN in a semiaggregate state inhibited cell proliferation. HUVEC Cell Counts For Aggregated and Semiaggregated Substrates After 7 Days of Being Seeded Cell Count for Cell Count for Cell Count for Substrate Control Semiaggregated Aggregated Surfaces* Surfaces Surfaces Gold 900 ± 17 716 ± 55 880 ± 17 Mica 908 ± 28 828 ± 22 940 ± 21 11-Mercapto- 289 ± 61 486 ± 25 851 ± 14 1-undecanol 11- 998 ± 10 826 ± 14 959 ± 8 Mercaptoundecanoi c acid 1-Hexadecanethiol 925 ± 12 738 ± 48 1088 ± 13

Average 804 ± 26 719 ± 33 944 ± 15

*Control surfaces reflect the amount of nonspecific adsorption when FN is absent from the substrate. Conclusion: Surfaces with aggregated FN promoted cellular deposition while surfaces with FN in a semiaggregate state appeared to hinder cellular deposition and growth Acknowledgments

™ We gratefully acknowledge William Wagner and Alexa Polk for providing the HUVEC cell culture. ™ Financial support for this work was provided by NSF-CRC (CHE-0404579), ARO-MURI (W911NF- 04-1-0191), and ONR (N00014-02-1-0327).

Some useful references: Meadows, P. Y.; Bemis, J. E.; Walker, G. C. “Single Molecule Force Spectroscopy of Isolated and Aggregated Fibronectin Proteins on Negatively Charged Surfaces in Aqueous Liquids”, Langmuir, 2003, 19, 9566-9572. Meadows, P. Y.; Walker, G. C. “Force Microscopy Studies of Fibronectin Adsorption and Subsequent Cellular Adhesion to Substrates with Well-Defined Surface Chemistries” Langmuir, ASAP, 2005. Meadows, P. Y.; Bemis, J. E., Walker, G. C. “Selective Interaction of Hydrophilic and Hydrophobic Blocks of Polystyrene-poly-2-vinylpyridine With a Silicon Nitride Surface Studied by Force Spectroscopy” J. Am. Chem. Soc., ASAP, 2005.