Molecular Interactions

The Surface Force Apparatus to Reveal the Energetics of Biomolecules

Assembly. Application to DNA Bases Pairing and SNARE Fusion

Proteins Folding

ERIC PEREZ,1 FENG LI,1 DAVID TARESTE,2 and FREDERIC PINCET1

1Laboratoire de Physique Statistique, UMR 8550 associee au CNRS et aux Universites Paris 6 et Paris 7, Ecole NormaleSuperieure, 24 rue Lhomond, 75005 Paris, France; and 2Department of Physiology and Cellular Biophysics,

Columbia University, 1150 Saint-Nicholas avenue, New York, NY 10032, USA

(Received 2 August 2008; accepted 19 September 2008; published online 10 October 2008)

Abstract—The Surface Force Apparatus (SFA) measuresdirectly, and with nanoscale resolution, the interaction energyvs. distance profile of planar arrays of biological molecules(e.g., lipids, polymers, or proteins). Through recent advancesin the reconstitution and deposition of lipid bilayers, it is nowpossible to use SFA to study the interactions betweenmembrane-incorporated biomolecules and to reveal anyconformational changes and intermediate assembly states.Therein we describe two example systems. First, we show thatusing bilayers functionalized to carry DNA bases on theirlipid headgroups, we can measure a macroscopic nucleoside–nucleoside adhesion force, from which one can obtain amolecular binding energy. Second, we describe the use of theSFA to study the interaction between SNARE proteins,which are involved in most of intracellular fusion events.Membrane fusion occurs when SNARE proteins assemblebetween lipid bilayers in the form of SNAREpins. SFAmeasurements between SNAREs embedded in lipid bilayersallowed us to elucidate the energetics and dynamics ofSNAREpin folding, and to capture an intermediate bindingstate in SNAREpin assembly.

Keywords—Surface force apparatus, Interaction energy,

DNA bases, SNAREs, Membrane fusion.

INTRODUCTION

Every major process in a cell involves dynamicassemblies and/or disassemblies of biological mole-cules. There has always been a strong interest inquantifying these interactions but generally studieshave been restricted to bulk thermodynamical mea-surements. The advent of nanotechniques allowedexperimentalists to measure the interaction between

surfaces coated with molecules whose position, den-sity, and orientation were precisely controlled, andthus to obtain information on fundamental interac-tions and single bond energies.

The Surface Force Apparatus (SFA)10 was thepioneer scientific instrument to measure nanoscaleforces. It was originally designed to study colloidalinteractions, including steric, electrostatic, van derWaals, and solvation forces, and today, it can also beused to monitor the assembly of biomolecules in realtime. Other force measurement techniques include: (i)the atomic force microscope (AFM)2 which measuresrupture forces between the tip of a cantilever and asurface; (ii) the optical1 or magnetic6 tweezers thatexert and measure forces on microscopic beads using afocused laser beam or a magnetic field, respectively;(iii) the Biomembrane Force Probe (BFP)5 which givesthe force between a bead and a surface using a redblood cell as a spring with tunable stiffness; and (iv)the flow chamber that provides the rate of formationand dissociation of molecular bonds submitted to ahydrodynamic flux.18

The SFA technique stems from right after WorldWar II as David Tabor was studying frictional inter-actions between surfaces in the Cavendish laboratory.He had an industrial contract aimed at developingimproved windscreen wipers, which led him to mode-lize the interactions between rubber and glass in water.Using hemispherical rubber samples pressed against aflat glass surface, his team could follow the interactionby interferometry. When the rubber/glass interactionexperiments were performed in air, contact wasimmediately established and the rubber hemispherebecame flattened over some area even under zero(compression) force. Under negative (pulling) force,and up to the onset of separation, the contact area

Address correspondence to Eric Perez, Laboratoire de Physique

Statistique, UMR 8550 associee au CNRS et aux Universites Paris 6

et Paris 7, Ecole Normale Superieure, 24 rue Lhomond, 75005 Paris,

France. Electronic mail: perez@lps.ens.fr

Cellular and Molecular Bioengineering, Vol. 1, No. 4, December 2008 (� 2008) pp. 240–246

DOI: 10.1007/s12195-008-0025-7

1865-5025/08/1200-0240/0 � 2008 Biomedical Engineering Society

240

remained nonzero, suggesting that attractive forceswere operating between these two solid surfaces. Thisobservation led his team to develop the JKR theory13

for the adhesion between two solid bodies, whichpredicts that the pull off force F to separate adeformable sphere of radius R from a plane is equal to:

F ¼ 3pRWadh=2 ð1Þ

where Wadh is the adhesion free energy per unit area ofthe two surfaces.

When the rubber/glass interaction experiments wereperformed in water, the team could not observe anyadhesive contact between the rubber and the glass.Upon increasing the compression force, the rubberwould deform and the contact area would flatten, butno adhesion would be produced. It soon becameobvious to them that they were dealing with theso-called repulsive electrostatic double-layer forces.

At the same time, Samuel Tolansky was extensivelystudying the surface topography of crystals usingFringes of Equal Chromatic Order (FECO).29 FECOare obtained by passing a white light through a Fabry–Perot interferometer, and are extremely sensitive to thesurface topography down to the angstrom scale. Theyturned out to be also very useful for surface forcemeasurements, since they could give the inter-surfacedistance and the deformation of two interacting sur-faces with an unprecedented resolution.

In order to study surface forces with better-definedsubstrates, Tabor’s team used mica which can be easilycleaved into atomically smooth thin sheets over severalsquare centimeters. This allowed them to produce thefirst detailed measurements of van der Waals forces inthe normal and retarded regimes.24 One of Tabor’sstudents, named Jacob Israelachvili, adapted the origi-nal design of the SFA to operate in liquids, andimproved its mechanics. He also solved the complete setof equations of the FECO, therefore improving theaccuracy ofmultiple-beam interferometry andmaking itmore user-friendly.8 Notably, this allowed him to sub-stantially improve the measurements of van der Waalsforces.12 After this, Israelachvili and his co-workersstudied various inter-surface and intermolecular forces,such as DLVO forces (repulsive electrostatic double-layer forces and attractive van der Waals forces),10

hydration forces,11 forces between lipid bilayers,17 andforces between receptors and their ligands.7

Other groups, such as the one of Jacob Klein,focused on polymer interactions at interfaces.16 Theorydescribes several cases of steric forces generated bypolymer layers. Among them, the mushroom modeldescribes the interaction of surfaces bearing end-grafted molecules which do not overlap laterally.Entropic in nature, these repulsive interactions come

from the restriction of the number of conformationsthat the polymer can take due to the presence of theother surface3,9:

F Dð ÞR¼ 72pCkBTe

� DRg ð2Þ

where F is the force between two surfaces (radius ofcurvature R) at a distance D from each other, C is thesurface density of the polymers, and Rg is their radiusof gyration.

As we describe below, the combination of SFAtechnology and polymer theory can give rise to veryprecise characterization of the repulsion and adhesionbetween two layers of proteins.

DESCRIPTION OF THE SFA TECHNIQUE

The SFA is based on astute mechanics combinedwith powerful interferometry, which allow the mea-surement of force vs. distance profiles between molec-ularly smooth mica surfaces, and can resolve distancesof 0.1 nm and forces of 0.1 lN.

The force is measured with a bending spring having atypical stiffness of ~100 N/m. The apparatus contains atranslation device which moves the lower mica surface(connected to the force-measuring spring) toward theupper one (Fig. 1). It employs two or three systems ofdistance control with increasing sensitivity. A coarselevel, consisting of a micrometric translation, allows fora rapid positioning of the two surfaces (accuracy~1 lm). A finer level, using a differential spring system,has an accuracy of ~1 nm. It consists of a helical springpressing against a cantilever spring one thousand timesstiffer (Fig. 2). When the helical spring is pressed onemicron further, it bends the cantilever spring by onenanometer, and thus moves the lower mica by the samedistance. This already provides accuracy sufficient formost of surface force studies. Addition of a piezoelec-tric system can further improve the sensitivity, andallows for positioning to an accuracy of ~0.1 nm.

The distance is measured by multiple beam inter-ferometry through a Fabry–Perot formed by the twomica sheets (of equal thickness 2–5 lm), free of crystalsteps, and silvered on the backside (Fig. 3). When awhite light is sent to this interferometer, the variouswavelengths are reflected and transmitted across thedifferent interfaces, and produce a transmitted spec-trum made of Fringes of Equal Chromatic Order(FECO). For a planar interferometer, the fringesconsist of lines in the spectrometer. For a curvedinterferometer, the fringes are curved, as shown inFig. 3. These FECO give the distance between the twomicas with a resolution of 0.1 nm.

SFA to Reveal the Energetics of Biomolecules Assembly 241

The mica sheets (typical area of 8 9 8 mm2) areglued with a thermosetting epoxy onto two cylindricallenses, which are then arranged in a crossed-cylindergeometry. This geometry (equivalent to a sphere-planegeometry) makes the alignment easier, and enablestesting many different surface regions. Accordingto the Derjaguin approximation,4 the force, F(D),between these two curved surfaces is proportional tothe interaction free energy per unit area, E(D), betweentwo equivalent planar surfaces if the distance is muchsmaller than the radius of curvature, R, of the surfaces,and if the interaction energy decreases sufficientlyrapidly with distance (at least in 1/D2):

FðDÞ=R ¼ 2pEðDÞ ð3Þ

SFA measurements thus provide directly the inter-action free energy per unit area between two surfacesas a function of their separation distance.

The SFA remains a challenging technique. First ofall, it is not easy to cleave mica and obtain a few squarecentimeters of thin sheets free of steps, of dust parti-cles, and of mica flakes. Then, upon gluing the micaonto the cylindrical lenses and mounting the lenses intothe apparatus, care has to be taken to avoid dustparticles or any other contamination to reach thesurfaces. The liquids and the chemicals used during theexperiment have to be ultra-pure in order to reducesources of contamination. In addition, the SFA has tobe installed on a vibration-free table to avoid noise inthe force measurements, and in a thermostated roomto reduce thermal drifts.

Mica surfaces can be coated with various mole-cules of interest (lipids, polymers, proteins), whichmakes it possible to study the complete interactionprofile of these molecules using the SFA, and toidentify the nature of the forces involved. From themid-eighties on, several SFA studies of lipid bilayerinteractions have been reported which aimed atcharacterizing the fundamental forces between mem-branes9: van der Waals forces, double-layer forces,hydration forces, and undulation forces. Once thetechnique was routinely applicable to lipid bilayers, itwas fairly straightforward to use functionalizedlipid bilayers whose headgroups would carry thefunction to be studied,19,25–27 or to follow anystructure modification of two interacting lipid lay-ers.20 When applied to interacting proteins (whosesize is typically of a few nanometers), the angstromresolution of the technique can provide the moleculardetail of their assembly, including conformationalchange and passage through any intermediate bind-ing state.15,22

Cantilever spring

Helical spring

Leafspring

White light

Prism

Spectrometer

Interference fringes

F(D)/R = 2π E(D)

Opposingmica surfaces

Cantilever spring

Helical spring

Leafspring

White light

Prism

Spectrometer

Interference fringes

F(D)/R = 2π E(D)

Opposingmica surfaces

FIGURE 1. The Surface Force Apparatus (SFA).

Stiff cantileverLeaf spring

Flexible helical spring

±1µm

±1nm.

Stiffness ratio: 1000

FIGURE 2. The differential spring system.

PEREZ et al.242

SFA TO MEASURE THE ENERGY OF DNA

BASES PAIRING

The dynamic double helix structure of the DNAmolecule is stabilized by highly specific interactionsbetween complementary nucleotides: Adenine bondingwith Thymine, and Guanine bonding with Cytosine.We have used the SFA to measure the adhesionbetween surfaces coated with the nucleosides (nucleo-tides lacking phosphate groups) Adenosine (A) orThymidine (T). Two lipids were synthesized whoseheadgroups were, respectively, made of A and Tnucleosides. These lipids were deposited onto mono-layer-coated mica surfaces, with the Langmuir–Blodgett technique, at a film pressure sufficiently highto ensure close-packing of the lipid layers. In thisconfiguration, the nucleosides are perpendicular totheir surface and oriented toward the other surface(Fig. 4); they are thus fully accessible for binding totheir complementary group residing on the oppositesurface.

Experiments were performed with symmetrical(A/A and T/T) and complementary (A/T) surface pairs(Fig. 4). The adhesion measured upon separating twocomplementary surfaces (A/T experiments) was morethan twice the adhesion of the symmetrical systems(A/A or T/T experiments). At contact, the surfaceswere flattened, and therefore the JKR theory (Eq. 1)could be used to evaluate their adhesion free energy.As the number of molecules was known on each sur-face from the Langmuir–Blodgett deposition parame-ters, it was then straightforward to estimate the energy

of the molecular bonds: the adhesion free energy perunit area Wadh is equal to the density of bonds multi-plied by the molecular binding energy eb:

White light

Plane interferometer

Fringes of Equal ChromaticOrder (FECO) seen in the

spectrometerA1 eikz

A9 e-ikz

A2 eikµ1z A3 eikµ3z

A6 e-ikµ3z

A4 eikµ1z

A8 eikz

A5 e-ikµ1z A7 e-ikµ1z

Ag AgMica MicaDistance

Curved interferometer

FIGURE 3. Interferometer made of two silvered mica sheets of equal thickness with refractive index l1, separated by anothermedium of refractive index l3. A beam of incident light is transmitted to the right, after multiple reflections, as lines observable in aspectrometer. The wavelength of these lines (and the position in the spectrometer) varies when the distance between the surfacesis changed. Bottom: When the silvered mica sheets are curved, the fringes observed in the spectrometer are curved.

AA AAA A AAA AA AA A AAAAA

T TT T TT T T TT T TT TTT T

DMPE lipids

functionalized lipids D

FIGURE 4. Interaction between DNA bases measured bySFA. The mica surfaces are coated with bilayers functional-ized to carry nucleosides on their lipid headgroups. Theadhesion measured in the complementary system (A/T) ismuch larger than for the symmetrical systems (A/A and T/T).

SFA to Reveal the Energetics of Biomolecules Assembly 243

Wadh ¼ density of bonds� eb ð4Þ

As the lipid layers are close-packed, every nucle-oside faces a nucleoside from the other surface, andone could assume that all molecules can form abond. However, because the binding energy of themolecules is small, two molecules which face eachother at atomic distance do not necessarily form abond. The probability that they form a bond isgiven by Boltzmann statistics: two interacting mole-cules can be either (i) unbound and in a zero energystate, or (ii) bound and in a -eb energy state. Thepartition function Z of the system is then: Z =1 + exp(eb), with eb expressed in thermal energyunits kBT, and the probability pbound that a bondforms is:

pbound ¼1

1þ expð�ebÞð5Þ

The energy of bond formation, deduced fromEqs. 1, 4 and 5, is 4.0 kBT (2.4 kcal/mol) for A/T, 2.2kBT (1.3 kcal/mol) for A/A, and 1.8 kBT (1.1 kcal/mol) for T/T.19,27 These values are in very goodagreement with those previously obtained fromquantum mechanics calculations21 or thermodynamicmeasurements.28

This result was followed by several similar studiesusing various functionalized lipid bilayers25–27 (e.g.,bilayers bearing chelating groups or groups that caninteract via a combination of hydrophobic forces andhydrogen bonds). In the case of chelation bonds, threeindependent methods were used to estimate the bind-ing energy (SFA, giant vesicle adhesion measurementsand microcalorimetry), which gave similar results.25 Itthus confirms that from a macroscopic adhesive forcemeasured by SFA, one can reliably deduce molecularbinding energies. In addition, it shows that theSFA is a powerful tool to quantify the interactionsof biomolecules in aqueous solutions, i.e. under close-to-native conditions.

SFA TO MONITOR SNARE FUSION PROTEINS

ASSOCIATION IN REAL TIME

Membrane fusion is a fundamental biological pro-cess which plays a central role in many areas ofphysiology, such as neuronal communication, viralinfection, or mitochondrial dynamics. Because bio-logical membranes are designed to be stable, mem-brane fusion does not occur spontaneously andrequires the intervention of specialized proteins whichprovide the energy required for fusion to occur.Intracellular trafficking events are governed by theassembly of cognate vesicular (v-) and target (t-)

SNARE proteins, which initially reside in apposingmembranes, to form a stable bridging complex (theSNAREpin) that triggers the membranes to merge.23,30

How much energy is made available from theSNAREpin protein-folding event, and how this energyis transmitted to the membranes destined to fuse arestill very much open questions.

Through SFA interaction energy measure-ments between membrane-embedded cognate v- andt-SNARE proteins, we have recently provided acomprehensive description in real time of the energet-ics and dynamics of SNAREpin folding acrossmembranes.15 The cytoplasmic domains of v- andt-SNARE proteins were modified to have only a singleCysteine residue at their C-terminal end, and thenchemically coupled through this Cysteine to a sup-ported lipid bilayer consisting of 89 mol% of DOPClipids, 10 mol% of negatively charged DOPS lipids,and 1 mol% of reacting DOPE-Maleimide lipids(Fig. 5). We tested many different pairs of SNAREbilayers, probing various regions on each of thesepairs. Each SFA experiment consisted of severalapproaching/separation cycles during which distancesand forces of interaction were measured simulta-neously every 30 s.

During the approaching phase, and before they startassociating (region between 20 nm and 8 nm in Fig. 5),SNARE proteins repelled each other like randompolymers. Upon separating two SNARE bilayers, astrong adhesion was systematically observed. Themeasured adhesive forces/radius varied however sub-stantially from one experiment to another (from 3 mN/m to 20 mN/m). Because we noticed a correlationbetween stronger adhesive forces and larger long-rangesteric repulsion, these variations were attributed todifferences in local SNARE density. The surface den-sity of SNARE proteins was estimated by modeling thelong-range repulsion experienced by SNARE bilayerswith the mushroom model of steric forces betweenpolymer layers (Eq. 1). Knowing the SNARE densityand the macroscopic adhesion energy of two cognateSNARE bilayers, we were thus able to deduce theSNAREpin binding energy: 35 kBT (21 kcal/mol),which corresponds closely to the energy needed tocreate the highly curved non-bilayer transition struc-tures (stalks) leading to fusion (40–50 kBT

14). By thesemeasures, the cooperative effect of a few SNAREpinsat the site of fusion would therefore release enoughenergy to overcome the high energetic barriers ofmembrane merging.

The force vs. distance profiles of assemblingSNAREpins also revealed that SNARE motifs beginto interact when the membranes are 8 nm apart, andthat the resulting SNAREpin is partially unstructuredin its C-terminal region (SNAREpin about 60–80%

PEREZ et al.244

zippered). In addition, membrane-proximal regions ofthe SNAREs (rich in basic amino acids) were shown tointeract with the negatively charged lipid membrane,which could contribute to partial SNARE assembly.Within a native fusion environment, such interactioncould play a role in functionally coupling SNAREassembly to the bending and fusion of two apposinglipid bilayers.

CONCLUSION

The Surface Force Apparatus is a powerful tech-nique to study in real time and in a close-to-nativeenvironment the interactions between biological mol-ecules. Its high resolution in distance and force allowsthe identification of any conformational changes andintermediate binding states that occur in the course oftheir assembly. In addition, as it measures the inter-action produced by a large number of identical mole-cules, the SFA enables averaging out any effect comingfrom thermal fluctuations, and thus makes it possibleto obtain directly the energetics of biomoleculesassembly, which single molecules techniques cannotcurrently do in a direct manner.

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