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 ‡ ‡ GΔ F() = Δ G −B Fx ⎛k h⎞ ‡ ⎜ off ⎟ ΔG = kB − ln T⎜ ⎟ ⎝kB T⎠ kB T 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 V(x,t) V= o (x)+ s − V (x vt) • led to the AFM’s piezo velocity, v. The Here, the reaction coordinate, x, is coup free energy is ⎧1 2 ⎫ k⎪ xm (x< β x⎪ ) ⎪2 ⎪ Vβ o (x)= ⎨ ⎬ −⎪ ∞(x ≥xβ ⎪ ) ⎩⎪ ⎭⎪ -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 e stat projected along the direction of applied force. Since it is e hassumed that t he fron on tsystem undergoes Brownian motiee energy surface, Kramers theory the relationship between the rateprovides of rupture in the absence of pulling (koff(0)) and the system’s properties through the equation below 2 3/ 2 ‡ k (x ) k ( )(x0 ;k≈ 2 )π −1Dk/ ( 2 ) x−BG Δe βΔG ‡ = m β off β m m β where 2 • Here D and ΔG‡ represent the diffusion coeffiding barrier cient and the unfol height, respectively. Rupture force vs. velocity, cont’d 1/ 2 kγ+ (x2 )2 / __ ⎡ m β ⎤ koff (0 )e Fβ = km β x2 −⎢ ln k ⎥, k= km + s k ⎢k vx (k3/ 2 /k)⎥ ⎣ 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.