surfacecharge differentiation of streptavidin and avidin by ......quot of vesicle solution was added...

DOI: 10.1002/cphc.201402234

Surface-Charge Differentiation of Streptavidin and Avidinby Atomic Force Microscopy–Force SpectroscopyLisa Almonte, Elena Lopez-Elvira, and Arturo M. Bar�*[a]

1. Introduction

Chemical analysis of matter consists of the determination of el-emental or molecular composition. The amount of material re-quired for chemical analysis has decreased continuously as in-creasingly sensitive analysis tools have become available. Theinvention of scanning tunnelling microscopy (STM) and relatedscanning probe microscopy methods with imaging capabilitiesdown to the atomic range,[1] has raised hopes that it may bepossible to determine atomic or molecular composition witha comparable resolution. In STM chemical recognition isachieved through spectroscopy of electronic states.

However, for non-conducting systems or for biological mate-rial that must be imaged in a liquid under physiological condi-tions, notable electronic states are not expected. Atomic forcemicroscopy (AFM) is a powerful technique for studying biologi-cal samples, owing to its ability to measure forces in real timeand in liquid media. Chemical analysis by AFM is done by forcespectroscopy (FS), which is based on the measurement offorce curves F(D) in the piconewton range, where F is the inter-action force between tip and specimen as a function of theirseparation D, measured in the nanometre range. Specific ornon-specific imaging is based on the differences in electrostat-ic, magnetic, optical or mechanical properties. Concerningtopographic imaging, frequency-modulation (FM) AFM imag-ing could be used to observe the surface topography of a lipidmembrane at the �ngstrçm scale.[2] Furthermore, the interac-tion of salts with lipid bilayers has been studied with molecularresolution.[3]

Although this paper deals exclusively with AFM–FS, methodsthat combine AFM with optical techniques such as optical fluo-rescence, confocal microscopy, total internal reflection fluores-cence microscopy, IR spectroscopy, near-field scanning opticalmicroscopy and tip-enhanced Raman scattering are also note-worthy.[4]

The aim of this work is to demonstrate that single moleculesof avidin and streptavidin deposited on a biotinylated bilayercan be differentiated by AFM, even though the two proteinsare indistinguishable in topographic images, because their pro-tein structures are almost identical. The strept(avidin)–biotinsystem [strept(avidin) denotes streptavidin or avidin] hasbecome a major mainstay in biochemical analysis and has far-reaching applications in biotechnology, industry and clinicalmedicine. The concept is to measure the different electrostaticinteractions of the two molecules with the AFM tip owing tothe different surface charges of avidin and streptavidin. Avidinis a basic glycoprotein (pI = 10.5), so that at pH 7 avidin is posi-tively charged,[5] whereas streptavidin is non-glycosylated witha near-neutral pI value.

Charging of a surface in a liquid can occur by various mech-anisms. Whatever the charging mechanism, the surface chargeis balanced by an equal and oppositely charged region ofcounterions, some of which are bound, usually transiently, inthe so-called Stern layer, whereas the others form a thin, dif-fuse charge cloud in rapid thermal motion close to the surface;together, they are known as the electric double layer (EDL).[6]

When two surfaces approach each other an interaction forcearises, owing to the overlap of their corresponding EDLs.

The basic physics is described by the Derjaguin–Landau–Verwey–Overbeck (DLVO) theory. The DLVO theory considerstwo contributions: the van der Waals attraction and the EDLforce. At large distances, the EDL force decays exponentially.The decay length is the so-called Debye length lD [Eq. (1)]:

Chemical information can be obtained by using atomic forcemicroscopy (AFM) and force spectroscopy (FS) with atomic ormolecular resolution, even in liquid media. The aim of thispaper is to demonstrate that single molecules of avidin andstreptavidin anchored to a biotinylated bilayer can be differen-tiated by using AFM, even though AFM topographical imagesof the two proteins are remarkably alike. At physiological pH,the basic glycoprotein avidin is positively charged, whereasstreptavidin is a neutral protein. This charge difference can be

determined with AFM, which can probe electrostatic double-layer forces by using FS. The force curves, owing to the elec-trostatic interaction, show major differences when measuredon top of each protein as well as on the lipid substrate. FSdata show that the two proteins are negatively charged. Nev-ertheless, avidin and streptavidin can be clearly distinguished,thus demonstrating the sensitivity of AFM to detect smallchanges in the charge state of macromolecules.

[a] L. Almonte, E. Lopez-Elvira, Prof. Dr. A. M. Bar�Department of Surfaces and CoatingsInstituto de Ciencia de Materiales de Madrid (CSIC)C/Sor Juana In�s de la Cruz 3, Campus de Cantoblanco (Spain)Fax: (+) 913720623E-mail : [email protected]

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2014, 15, 2768 – 2773 2768

CHEMPHYSCHEMARTICLES

lD ¼ee0kBT

e2P

i

ciz2i

0

@

1

A

1=2

ð1Þ

where e is the dielectric constant of the liquid, e0 is thevacuum permittivity, kBT the Boltzmann factor, e is the elemen-tary charge, and ci and zi are the concentration and valence ofthe ions in the solution.

In continuum theory the potential distribution is determinedfrom the Poisson–Boltzmann equation, which is a second-orderdifferential equation. The force between a parabolic AFM tipwith a radius of R and a flat surface can be obtained for con-stant-charge conditions, which apply in AFM.[7] Further approx-imation leads to a very simple equation [Eq. (2)] that is onlyvalid under several assumptions, including small surface poten-tials, tip–sample separations D larger than the Debye length lDand tip radii larger than the separation (R @ D�lD):

FEDLðDÞ ¼4 pRstipssamplelD

ee0exp� D

lDð2Þ

where stip and ssample are the surface charge densities of tipand sample.

Equation (2) permits ssample to be determined by measuringthe force curves FEDL(D) as a function of D. Indeed FEDL(D) isproportional to ssample. Therefore, by measuring FEDL(D) foravidin and streptavidin we can obtain the different surfacecharges of the two proteins. This is in fact a particular case ofchemical analysis of streptavidin and avidin, because the sur-face charge is due to the protein composition.

Experimental Section

Materials

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DOPC), avidin (fromegg white), streptavidin (from Streptomyces avidinii) and phos-phate-buffered saline (PBS) were purchased from Sigma-Aldrich.1,2-dipalmitoyl-sn-glycero-3 phospho-ethanolamine-N-(biotinyl) (b-DPPE), purchased from Avanti Polar Lipids, Inc. and Milli-Q water,deionised to a resistivity of 18 MW cm, were used in the experi-ments. Avidin and streptavidin are both composed of four identicalsubunits, each of which binds one biotin molecule.

Supported lipids bilayers (SLBs) are currently used as supports foranchoring biomolecules because they are used as model mem-branes to study protein binding to lipid ligands and membrane in-sertion of proteins.[8] Properties of lipid bilayer membranes andmembrane–protein complexes have been of great interest and thesubject of numerous studies, owing to their physical and biologicalimportance and potential applications. We employed b-DPPE/DOPC bilayers as supports. In previous reports,[9–11] AFM was usedto investigate a specific recognition reaction: the binding of strept-(avidin) to a biotinylated bilayer. Even after the transfer of a biotiny-lated bilayer to a solid support, the biotin receptor remained ac-cessible and reactive to strept(avidin).

A DOPC/b-DPPE mixture (9.9/0.1 mol/mol) was dissolved in chloro-form to give a final concentration of 0.7 mg mL�1. The solution wasevaporated in a nitrogen flow to leave a film at the bottom of thevial. The lipid film was hydrated in a buffer solution (10 mm phos-

phate, 137 mm NaCl, 27 mm KCl, pH 7.40 at 25 8C). The vials weresubjected to several cycles of vortexing, and then repeatedly ex-truded through polycarbonate membranes with well-defined poresto obtain 200 nm unilamellar vesicles.

SLBs were obtained by using the vesicle fusion method.[12] An ali-quot of vesicle solution was added to freshly cleaved mica and themixture was incubated for 2 h. Subsequently, the sample wasrinsed with buffer. Strept(avidin) was released in SLB to a concentra-tion of 1.25 mg mL�1, incubated for 45 min at 25 8C, and thenwashed to remove proteins that were not anchored to the sup-port. Then the tip was immediately immersed into the sample formeasurements. With this incubation time and 1 % of the biotinylat-ed lipid, a low coverage of proteins is obtained, and thus single-molecule studies are possible.

Methods

AFM measurements were performed in liquid medium at roomtemperature by using a commercial set-up (Nanotec S.L. , TresCantos, Spain), controlled with the WSxM software.[13] Rectangularsilicon nitride cantilever tips with a nominal spring constant of50 pN nm�1 were employed (OMLC-RC800PSA-W, Olympus, Japan).For silicon nitride tips, it is accepted that pI

method is based on the analysis of the F(D) curve. At largetip–sample distances, the force is zero, but when this distancedecreases, the force grows, slowly at first, until the tip comesinto contact with the bilayer; further indentation results ina vertical increase in force, whereby the distance remains con-stant. The sample withstands the force without deformationprior to rupture of the bilayer and the tip impinging on themica substrate. The resulting well-defined step in the forcecurve allows the bilayer thickness (�6 nm) and the breakingforce of the bilayer (�2.7 nN) to be measured. Figure 1 bshows an AFM topography image of an incomplete SLB coat-ing on mica obtained in the jumping mode.[22] The height ofthe lipid bilayer measured from a profile (Figure 1 c) coincideswith that obtained from the curve in Figure 1 a.

2.2. Application of the FSI Mode to Strept(avidin) Supportedon the Biotinylated Bilayer

The FSI method was used to obtain single-molecule resolution.Raw data from an FSI file consists of a topographic image anda force curve associated with each pixel of the probed surfacearea. In this experiment an FSI file contained 64 � 64 pixels.Raw force curves must be represented in terms of force versusreal tip–sample distance for further analysis, so automatic dataprocessing is mandatory. Recently, a simplified model for quan-titatively studying EDL forces on single adsorbed moleculeswas proposed.[23] In this model the EDL force on top of thesubstrate and on top of the molecules can be described bya function that decays exponentially with the tip–substrate dis-tance (dts) [Eq. (3)]:

FEDLðdtsÞ ¼ FT0 expð�dts=lDÞ ð3Þ

where FT0 is the amplitude of the total force. The fitting of theforce curves to the force law is performed by a least-squaresmethod. From the fits, the amplitude and decay length of theforce are obtained.

Owing to the presence of the molecules, dts changes; there-fore, it is necessary to transform force curves into a representa-tive force versus tip–sample distance [F(dts)] . This is done byshifting the contact point of the experimentally measuredforce versus tip–sample distance curves by the correspondingheight of the topography image (assuming zero height for thesubstrate). In other words, from the top of the avidin moleculedtm = dts�hm, where hm is the molecular height (Figure 2).

If we assume that the EDL force dominates the tip–sampleinteraction, two parameters, FT0 and lD, can be obtained by fit-ting F(dts) curves to exponential functions [Eq. (3)] . From theclean substrate, the molecules do not contribute to the forceexerted on the tip. Thus, in this case the EDL force amplitudeFT0 is the same as that of a pure tip–substrate interaction Fts0.From a molecule, however, FT0 is the result of two contribu-tions: the tip–substrate force amplitude Fts0 and the EDL forcebetween the tip and the molecule Ftm0.

According to the force amplitude from Equation (2), force isproportional to the surface charge densities of the probe stand a molecule sm. Hence, for similar tip–substrate separations,EDL forces should be more repulsive near a molecule than farfrom it, if the molecule has the same surface-charge sign asthe probe. In contrast, if it has the opposite sign, an attractiveforce should be observed.

2.3. FSI Files of Avidin and Streptavidin Specimens

Once we checked that the biotinylated bilayer was formed,two different (strept)avidin specimens were prepared .The firstwas prepared by dropping an avidin solution with a concentra-tion of 1.25 mg mL�1 and incubation time of 45 min (sample A),and the second one by dropping a streptavidin solution (sam-ple B) under the same conditions. Representative topographiesof samples A and B are shown Figure 3 a, b. These were ob-tained from a measured FSI file, which provided F(D) curves foreach pixel (x,y). For a specific pixel (x0,y0), D0 (contact point)corresponds to F(D0) = Freference, which is identical to the topog-raphy. The strept(avidin) proteins are bumps of about 4–5 nmin height. The width is larger, owing to tip dilation.[7] We attri-bute the variation in the size of the proteins to the formationof aggregates (when the size is larger than the height of the

Figure 1. a) Force–distance curve showing the indentation of the lipid bilay-er. The bilayer breaks at a breaking force of 2.7 nN, and the bilayer thicknessis 6 nm. b) AFM topography image of an incomplete SLB coating, in whichthe mica substrate can be seen. c) Profile of SLB on mica corresponding tothe grey line in b). The height of the profile is about 6 nm, similar to thevalue obtained in a).

Figure 2. Schematic representation of the sample showing strept(avidin)anchored to biotinylated SLB supported on mica. The main geometrical pa-rameters employed to obtain the EDL force are shown.


CHEMPHYSCHEMARTICLES www.chemphyschem.org

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protein) and to the rupture of some protein molecules in thefreezing and thawing process (when the size is larger than theexpected height of the protein).

Representative F(dts) curves for samples A and B are shownin Figure 3 c, d. F(dts) curves on top of the bilayer were mea-sured on both samples A and B; they can be used as referen-ces for force curves of sample A from the top of avidin (Fig-ure 3 c) and sample B from the top of streptavidin (Figure 3 d).Furthermore, the avidin and streptavidin curves are shiftedwith respect to the bilayer curves by the protein height hm�4.5 nm. (dts = dtm + hm). From the fitting of these curves toF(dts) exponential functions (see Section 3.2), we obtained FTO =(292�33) pN and lD = (2.9�0.5) nm for streptavidin, and FTO =(77�14) pN and lD = (3.4�0.9) nm for avidin. Herein, thesemagnitudes are presented as mean values calculated by aver-aging those obtained by fitting several curves measured atsimilar positions (i.e. the top of the SLB or the top of the twoproteins). The R2 value provides information about the quality

of the fit. The values obtainedwere R2�0.93 for avidin and R2�0.95 for streptavidin.

Iso-distance maps are an illus-trative way of mapping the dis-tribution of EDL forces on sur-faces. These maps show how thepresence of the molecules af-fects the tip–substrate interac-tion. They are built from FSImeasurements by representing,for each point of the sample, thenormal force at a given distancefrom a base plane, which in thecase of adsorbed molecules isusually chosen to be that de-fined by the substrate.[19] Theiso-distance maps (Figure 3 e, f)were obtained by using thesame FSI image as in Fig-ure 3 a, b. They were taken atdts = 7 nm (a different distancecould be used as well, but atlonger distances the force is toolow and noisy, and at shorterdistances the FSI image is tooclose to the topography for EDLdistinction of the two proteins).At dts = 7 nm, the EDL map ofproteins is brighter than that ofthe SLB substrate (Figure 3 e, f),in agreement with the F(D)curves shown in Figure 3 c, d.The streptavidin molecules inthe iso-distance map havehigher contrast, whereas foravidin molecules the contrast islower. To avoid this problem we

scaled the z axis to give the same brightness for both proteins,as FEDL(avidin)

The EDL forces for streptavidin in Figure 4 b were obtainedas averages of the FT0 curve values for streptavidin moleculesfor each corresponding FSI file (samples 1, 2 and 3). The sameprocess was carried out for samples of avidin on the SLB (sam-ples 4, 5 and 6). The values obtained for the SLB are similar inthe experiments on samples A and B. In Figure 4 b the normal-ised FT0 values vary from 15.48 to 17.77 pN nm

�1 for streptavi-din, from 4.66 to 5.17 pN nm�1 for avidin and from 2.34 to2.84 pN nm�1 for the SLB. The variations in values may be dueto slight changes in the charge density of the tip (e.g. impuri-ties adhering to the tip).

3. Discussion

It is important to understand how AFM–FS is capable of dis-criminating between avidin and streptavidin. The concept isthat each surface immersed in an electrolyte develops a surfacecharge, and hence an EDL. When two surfaces have differentsurface charges they can be differentiated. The force is verysensitive because it is proportional to the surface charge densi-ty of the sample.

From Equation (2), the EDL interaction force F between tipand sample is a function of its distance D and proportional tosurface charge densities stip and ssample. The Debye length lD isanother important quantity and depends according to Equa-

tion (1) on the concentration and valence of the ions as well asthe pH of the buffer solution.

A silicon nitride tip is negatively charged, owing to its pIvalue of less than 6 at a solution pH of 7.4.[14] As the EDLforces are all repulsive for the lipid, avidin and streptavidin(Figure 3 c, d), we deduce that all three are negatively charged.In addition, taking into account the EDL forces of lipid andproteins, the charges are related by the inequalities jslipid j < jsavidin j < jsstreptavidin j . This inequality permits avidin and strepta-vidin to be discriminated, as shown in Figure 4, although thesame signs of s for the two homologous proteins makes thedifferentiation more demanding. At the same time, this showsthat the EDL force method has a high sensitivity.

We now discuss the charge signs of the three specimens(the bilayer, streptavidin and avidin). We start by analysing thecase of zwitterionic lipids (our case), because we think thatthey have a non-negligible influence on the electrical proper-ties of the proteins. Zwitterionic lipids should be neutral, butthey are negatively charged at physiological pH. We alsoexpect that the membrane electrostatic potentials change thelocal concentration of ions and small molecules.[25]

The slightly repulsive force curve on the SLB (44 pN) indi-cates that the bilayer has a tiny negative charge. A similarresult has been reported for the analysis of pure zwitterionicmembranes of DOPC by AFM with a negatively charged siliconnitride tip. The repulsive measured force curves indicated thatthe zwitterionic membrane was negatively charged.[26] Yanget al. attributed the repulsive interaction to counterion bind-ing. This could also be applied to our case, that is, the zwitter-ionic membrane attains negative surface charge, owing tocounterion binding (our buffer solution contains 137 mm NaCland 27 mm KCl). As an interesting alternative, Yang et al. pro-posed that the repulsive interaction is due to an external ves-tige of the large internal electrostatic fields associated with thedipole potential between the head-group and hydrophobic re-gions of the membrane. We think that the dipole potentialcould also be the origin of the negative charge of the biotiny-lated bilayer.

Now we consider the case of (strept)avidin specificallybound to biotin by direct ligand association. Electrostatic andvan der Waals forces were found to dominate the long-rangenon-specific interaction at >2 nm.[27] Some investigations illus-trate the importance and continued interest in determiningthe impact of electrostatics on molecular recognition. The EDLforce between two streptavidin monolayers anchored to a sup-ported biotinylated bilayer was measured with a surface-forceapparatus, and the pH dependence of the charge density ofstreptavidin was reported.[28] The directly measured isoelectricpoint of 5.0�0.5 for the biotin binding obtained with thismethod differs from the near-neutral pI value of the protein insolution. This demonstrates that the electrostatic forces reflectthe local charge density of the exposed protein surface. In thecase of streptavidin, the measured isoelectric point increasesthe negative charge (at pH 7.4) for the biotin binding, whichqualitatively explains the observed negative charge for strept-avidin even though it is a neutral homologue of avidin. Wethink that the same situation probably occurs with avidin and

Figure 4. a) Merged force versus tip–molecule distance curves on top of pro-teins obtained from six different FSI files [three for sample A (avidin) andthree for sample B (streptavidin)] . b) Histogram of forces normalised by theradius of the tip, obtained from the same six FSI files as in a) for streptavidin,avidin, and the SLB in the corresponding experiments. The bars in the col-umns show the error for each data set.



www.chemphyschem.org

leads to a lower pI value for avidin, but we did not find any re-ported experiment in this context in the literature. On theother hand, assuming that the avidin–biotin binding still haspositive charge (pI>7.4), a charge inversion may take place onthe complex, owing to charge screening produced by the neg-ative ions located around.

Finally, from Equation (2) the experimental charge densitiesof the molecules and the lipid were obtained, whereby FEDLwas calculated from Equation (3). The values obtained aresstreptavidin =�33�6.3 mC m�2, savidin =�6�0.3 mC m�2 andsSLB =�4�1.4 mC m�2. These values are averages obtainedfrom the three experiments shown in Figure 4. In ref. [28]a value of sstreptavidin =�19 mC m�2 at pH 7.6 in 6 mm phos-phate buffer is reported. This is in agreement with the valuewe obtained, because in our case the concentration of ions ishigher (10 mm) and thus a more negative value of the chargedensity is expected.

Conclusions

The different electrostatic interactions of streptavidin andavidin on a biotinylated bilayer support was shown by measur-ing the laterally resolved EDL force by AFM–FS. This permitsdifferentiation of the two homologous proteins despite theirindistinguishable topographic images, and thus chemical infor-mation can be obtained. The unexpected values of the surfacecharge density of strept(avidin) are attributed to the ions inthe buffer and the complex interaction of the two proteinswith the bilayer support. Finally, we stress the importance ofusing AFM, which can characterise the local electrostatic prop-erties of the lipid–protein interaction.

Acknowledgements

We acknowledge funding by the MICINN through projectsFIS2012-35248 and CSD2009-00024 and by the CAM throughproject Nanoobjetos (S2009/MAT-1467).

Keywords: bilayers · force spectroscopy · lipids · proteins ·scanning probe microscopy

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Received: April 11, 2014

Published online on July 2, 2014


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surfacecharge differentiation of streptavidin and avidin by ......quot of vesicle solution was added...

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