Pretransition and progressive softening of bovine

carbonic anhydrase II as probed by singlemolecule atomic force microscopy

REHANA AFRIN, MOHAMMAD T. ALAM, AND ATSUSHI IKAILaboratory of Biodynamics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,Midori-ku, Yokohama, 226-8501, Japan

(RECEIVED December 8, 2004; FINAL REVISION March 6, 2005; ACCEPTED March 11, 2005)

Abstract

To develop a simple method for probing the physical state of surface adsorbed proteins, we adoptedthe force curve mode of an atomic force microscope (AFM) to extract information on the mechanicalproperties of surface immobilized bovine carbonic anhydrase II under native conditions and in thecourse of guanidinium chloride–induced denaturation. A progressive increase in the population ofindividually softened molecules was probed under mildly to fully denaturing conditions. The use ofthe approach regime of force curves gave information regarding the height and rigidity of themolecule under compressive stress, whereas use of the retracting regime of the curves gave informa-tion about the tensile characteristics of the protein. The results showed that protein molecules at thebeginning of the transition region possessed slightly more flattened and significantly more softenedconformations compared with that of native molecules, but were still not fully denatured, in agree-ment with results based on solution studies. Thus the force curve mode of an AFM was shown to besensitive enough to provide information concerning the different physical states of single molecules ofglobular proteins.

Keywords: bovine carbonic anhydrase II; atomic force microscopy (AFM); protein stretching andcompression; Young’s modulus; progressive softening; pretransition softening; Tatara model

The atomic force microscope (AFM) is widely used tocharacterize biomacromolecules such as proteins, polysac-charides, andDNAat the singlemolecular level; providing awealth of knowledge on their mechanical properties by thedirect application of compressive or tensile force to individ-ual molecules (Radmacher et al. 1994; Mitsui et al. 1996;Rief et al. 1997; Carrion-Vazquez et al. 1999; Clausen-Schaumann et al. 2000; Li et al. 2000; Wang et al. 2001;Alam et al. 2002; Hertadi et al. 2003; Moller et al. 2003). Inmost of the studies cited above, macromolecules were

stretched by the application of tensile forces in opposingdirections fromtwoarbitrarily chosenor specificallydefinedpoints along their primary structures, providing informa-tionon the internal heterogeneity in theirmechanical design.In just a limited number of cases has an AFM been used tocompress protein molecules to extract information on theapparent Young’s modulus of individual molecules; forexample, in the case of lysozyme, it was reported to bein the range of 0.56 0.2 GPa (Radmacher et al. 1994).Although the actual measurement of Young’s modulususing either an AFM or a surface force apparatus has sofar given values in a 0.2 GPa to 2 GPa range (Morozov andMorozova 1981; Radmacher et al. 1994; Suda et. al. 1995 ),a theoretical assessment of protein rigidity has recently beenput forward proposing 0.05–0.5GPa as a plausible range ofthe mechanical modulus of protein molecules (Vanselow2002).

ps0412823 Afrin et al. Article RA

Reprint requests to: Atsushi Ikai, Laboratory of Biodynamics,Graduate School of Bioscience and Biotechnology, Tokyo Instituteof Technology, 4259 Nagatsuka, Midori-ku, Yokohama, 226-8501,Japan; e-mail: aikai@bio.titech.ac.jp; fax: 81-45-924-5806.Article and publication are at http://www.proteinscience.org/cgi/

doi/10.1110/ps.041282305.

Protein Science (2005), 14:1447–1457. Published by Cold Spring Harbor Laboratory Press. Copyright ª 2005 The Protein Society 1447

Development of a method to evaluate protein rigidityand, if possible, local variations of it is importantbecause protein flexibility has been recognized as anessential requirement in the expression of enzyme activ-ity, muscle contraction, ligand–receptor interactions,and many other protein functions (Cantor andSchimmel 1980). It has also been studied using differentexperimental techniques such as pressure-dependentNMR spectroscopy or acoustic measurement of thecompressibility of proteins in solution (Gekko 2002;Akasaka 2003). Further, the physical state of surfaceadsorbed protein has been an important issue in thefield of surface science (Karlsson et al. 2000; Ramsden2002; Vermeer 2002) and biotechnology (Wahlgren andArnebrant 1991; Elwing 1998; Tengvall et al. 1998;Drobny et al. 2003). Atomic force microscopy has thepotential to make significant contributions to the studyof protein flexibility by allowing researchers to press and/or pull single protein molecules in specified directions.

Bovine carbonic anhydrase II (BCA II) is a zincmetalloprotein with 259 amino acid residues and isknown to undergo a transition from the native to a fullydenatured state via a stable intermediate state(s) when theconcentration of guanidinium chloride (GdmCl) isincreased from 0 M to 6 M (Wong and Tanford 1973).Characterization of the pretransition intermediate statesof the protein has been actively pursued for bovine as wellas human CA by using a variety of optical, hydrodynami-cal, spin-labeling, and chromatographymethods. It is nowwell accepted that the pretransition state of this proteinunder mildly denaturing conditions has properties ofwhich many can be ascribed to the ‘‘molten globule’’state of the protein (Uversky 1993; Boren et al. 1999;Carlsson and Johnsson 2000; Andersson et al. 2001). Inthe molten globule state, the hydrodynamics radius of theprotein is similar to that of the native protein, but mostof the experimental parameters indicate that the proteinis devoid of the segmental interactions required for keep-ing its well-defined three-dimensional conformation,although the optical parameters strongly support thatthe secondary structure of the protein is formed almostto the same level as the native protein. The proposednature of the molten globule-like intermediate stateof BCA II presents itself as an ideal test case for applica-tion of AFM-based mechanical measurement at thesingle molecular level. We previously reported themechanical response of the recombinantly modifiedprotein with two cysteine residues outside of N andC termini under native and fully denatured conditionsusing the force curve mode of an AFM (Wang et al.2001). In this report we investigate whether an AFM-based mechanical method can distinguish the pretransi-tion intermediate state from either the native or fullydenatured states.

Results

Results under native conditions

Figure 1 gives a schematic view of the force modeoperation of the AFM with a BCA II molecule sand-wiched between the tip and the substrate. The radiusof the tip is shown as several times larger than that ofthe sample protein, which is close to the actual ratio ofthe two radii. The three circles in black dotted lineindicate the positions of alternate crossings of theC- and N-terminal segments together forming a ‘‘knotstructure.’’

In Figure 2A, an example of the raw output from theAFM is given to explain the data acquisition method.In this figure, the abscissa is the distance between thesubstrate and the tip, and the ordinate is the deflectionof the cantilever with positive values meaning upwarddeflection. The AFM tip first starts from point 1,approaching the sample surface, and proceeds horizon-tally to point 2, where the curve starts to climb with anincreasing curvature from zero. The curve asymptotic-ally approaches the dotted line with a slope of �1,implying, first, a contact of the tip to the sample proteinat point 2 followed by a gradual upward deflection ofthe cantilever due to compression of the soft sample. Ifthe sample surface is undeformable, the curve will be astraight line with a slope of �1 from point 2 up (dashedline). The sample stage movement was then reversed atpoint 3 and tip-sample contact broken at point 4, butwhen a tensile molecule is sandwiched between the tip

Figure 1. A schematic view of force curve operation of the AFM with

a bovine carbonic anhydrase molecule sandwiched between the AFM

tip and the substrate through covalent cross-linkers. Three alternative

crossings of the chain in the C-terminal region leading to the knot

formation are indicated with circles in black dotted lines.

Fig 1. live 4/c

1448 Protein Science, vol. 14

Afrin et al.

and the substrate, a downward deflection of the canti-lever was recorded up to point 5 with a negative canti-lever deflection. At point 5, the cantilever is released tothe level position 6 after rupture of the bond betweenthe sample and the tip, and then retracted back to theoriginal position at point 1. The red letters in italics, D,d, E, and I, in the figure represent the tip-substrate

distance, the deflection of the cantilever, the extensionof the sample, and the depth of compression (or inden-tation), respectively. Conversion of the raw force curvedata into force extension (F–E) curves was done byusing the relations F¼�k3 d and E¼D� d, where alinear response of the cantilever against deflection wasassumed. The result of such conversion of the data inFigure 2A is given in Figure 2B, where the abscissa isthe chain elongation in nm and the ordinate is thetensile force in nN. In this example, the compressionof the molecule was reversible, implying that the mole-cule behaved as an elastic material with little plasticdeformation.

For protein immobilization, an aliquot of a proteinsolution was dropped onto a silicon surface modifiedwith APTES and LC-SPDP (for abbreviations, seeMethods and Materials), and left for 20 min for thecross-linking reaction to take place. After washing thesilicon substrate with a protein-free buffer solution,force curve measurement was started within 30 min ofthe exposure of the protein to each experimental condi-tion, and data were taken within the next 1–2 h. Ourpreliminary experiment showed that force curves taken>3 h after immobilization of the protein on a modifiedsilicon surface started to show indications of surfacedenaturation.

We first introduce in Figure 3A, the result of pullingexperiments on BCA II in a 50 mM Tris-sulfate buffer(pH 7.5) at �25�C. The inset figures give representativeindividual curves with initial extension of �15 nm (1) and�30 nm (2), respectively. A majority of the pulling curvesshowedan initial low force response (0.2–0.25nN) for�10–30 nm, and then the force increased rapidly without anysignificant further extension of the sample (without proteinsamples, force curves were almost flat, showing little or nointeractions).When the force reached the 1–2nN range, thecovalent cross-linking system was ruptured, and no tensileinteractions were obtained beyond the rupture point. Thisbehavior of BCA II in the early phase of forced stretching issimilar to the previously reported observation of Wanget al. (2001), who attributed this effect to the presence of a‘‘knot’’ structure in theC-terminal region of the protein (forBCA II, see Saito et al. 2004; forHCA II, see Eriksson et al.1988; Hakansson et al. 1992). There appear to be twogroups of force curves in Figure 3A with respect to thelength of the chain extension achieved before the covalentbond was ruptured: one in�15-nm range and the other in�30-nm range. It is thus possible that the protein can beunfolded in two different ways with respect to the initialknot tightening processes. Our preliminary result of molec-ular dynamics simulations of chain extension of knottedBCA II confirmed the appearance of large force peaksat �15 nm and �30 nm in extension, which explainsthe presence of �15 nm peak described above, but the

Figure 2. (A) An example of the raw output of the force curve mode of

AFM operation. The tip starts from position 1 and touches the sample at

position2, fromwhere the cantilever is pushedup toposition3.Thence, the

tip reverses its movement up to position 4, and the cantilever is pulled

downward toposition5due to thepresenceof tensile sample.Atposition5,

a covalent bondyields to the tensile force, and the cantilever jumps back to

its free position at 6. D, d, E, and I are, respectively, the distance between

the tip and the substrate, cantilever deflection, sample extension, and the

depthof compression.The total distance covered by the piezomotorunder

the sample stage isD¼E+d. (B) The rawdata above are converted to the

F–E curve by plotting the tensile force, F¼�k3 d versusE, where k is the

spring constant of the cantilever. The horizontal positions marked as

H and E0, respectively, indicate the contact positions of the tip to the

protein upon their mutual approach and the point of their final separation

upon retraction. They, therefore, respectively correspond to the approx-

imate height of the sample and the contour length of the protein, provided

that the chain is stretched normal to the substrate surface.

Fig 2. live 4/c

www.proteinscience.org 1449

Protein softening probed by AFM

disappearance of the same peak in �30 nm extensionremains to be explained.

Even under native conditions, we found the presence ofa fully stretchable population of BCA II without showingany significant force peaks until the final peak of covalentbond rupture, which indicated that a certain population ofthe protein (�20%) was nearly denatured. Since the activ-ity of the protein was �100% as measured before immo-bilization on a silicon surface, the apparent softening musthave occurred after immobilization.

When the immobilized protein was incubated with theinhibitor, p-aminomethylbenzenesulfonamide, in theAFM liquid cell and used for force curve measurements,

the F–E curve showed a slight change from those with-out the inhibitor, as shown in Figure 3B with an insetfigure showing representative individual curves here andhereafter. The initial extension before the rupture of thecovalent bond became longer by �5–10 nm comparedwith those in Figure 3A and the depth of compression isshown to be only 2 nm compared with 3.56 0.1 nm forthe native protein, indicating an increased rigidity of thecore structure accompanied by a slight loosening ofsurface residues of the protein as discussed later.

We next examine the compression part of the forcecurve. The part of the F–E curve in the contact region inFigure 4A is given as an overlap of 15 experimental curvesobtained under native conditions. In this figure, theabscissa, IH, is one half of the experimentally observeddepth of compression, I, in accordance with the theoreticalanalysis of contact mechanics as explained below. H/2 istherefore one half of the contact height of the sample. Sincethe actual dimension of BCA II is �63 43 4 nm (Saitoet al. 2004), the value ofH¼ 3.56 0.1 nm was slightly lessthan the expected height of the protein. The result impliedthat the protein is not completely flattened under the forceof 1–1.5 nN. The compression curve was reversibleapproximately up to 2 nm in the depth of compression.

In the following sections, we apply the classic Hertzmodel of contact deformation (Johnson 1985) and itsmodification by Tatara (1989, 1991; Tatara et al. 1991).The original Hertz model for a small deformation of ahalf-sphere of radius R1 under compressive contact witha spherical probe of radius R2 predicts that the force, F,depends on the 1.5th power of IH as in Equation 1 below(Johnson 1985). In the subsequent formulas, subscripts 1and 2, respectively, denote the quantities for the proteinmolecule and the tip, with common notations of Y and �,respectively, for Young’s modulus and Poisson’s ratio.

F ¼ 4

3

ffiffiffiffiR

pY1

ð1� �21ÞI

3=2H ¼ aI

3=2H

ð1Þ

where, R ¼ R1R2= R1 þ R2ð Þ and a ¼ 4

3

ffiffiffiffiR

pY1

1� � 21

� � :

When we apply the original Hertz model to obtainYoung’s modulus of the protein molecule, we mustremember that the model was derived for a semi-infiniteelastic sphere, i.e., a large half sphere in a simple expres-sion, assuming a small deformation at the contact site ofthe two spheres. In the analysis of compressive curves inFigure 4A, we considered four different possibilities, asshown schematically in the bottom part of Figure 4B. Incases 1 and 3, a protein molecule is compressed equallyfrom the top and bottom with a small (1) or large (3)total deformation. The measured value of I in these casesmust be halved to fit the experimental data to the

Figure 3. (A) The F–E curves of BCA II under native conditions with

50 mM Tris-sulfate buffer (pH 7.5) and at �25�C. The initial low level

forces in the range of 0.1–0.2 nN are considered to represent frictional

resistance in the C-terminal knotted region. Insets 1 and 2 contain

representative individual curves with �15 nm and �30 nm initial

extensions, respectively. (B) F–E curve obtained in the presence of

the inhibitor. Inset is a collection of representative individual curves.

1450 Protein Science, vol. 14

Afrin et al.

theoretical curves because the Hertzian approach dis-tance, IH, used in theoretical models represents thedepth of compression at a single contact site; in cases2 and 4, a protein molecule is immobilized on the sub-strate with a large contact area compared with its contactsite with the tip. In these cases, at least in the initialperiod of compression, the measured I can be taken as

IH and is applicable to denatured protein molecules onthe substrate (‘‘mushrooms’’ as later introduced).

Since Hertz model predicts 1.5th-order dependence ofF on the Hertzian approach distance (IH) (Johnson1985), we first applied Equation 2 with a variableYoung’s modulus, Yapp, where Y0 and k are adjustableparameters, to reproduce the experimental curves forthe initial estimation of the value of a:

F ¼ 4

3

ffiffiffiffiR

pYapp

1� � 21

� � I 3=2H ð2Þ

where, Yapp ¼ Y0½1þ k exp IHð Þ�:The resulting values of apparent Young’s modulus

Yapp are plotted in Figure 5 as a function of IH. Theresult indicates that for substantially denatured proteins(curves 4, 5, and 6), Yapp is almost constant and repre-sents the Young’s modulus of the samples correctly, butfor curves 1, 2, and possibly 3, we need more advancedanalyses than the classical Hertz model.

For a more precise analysis of compression curve ofnative BCA II in Figure 4A, we applied the originalHertz model (Equation 1), Tatara model (Tatara 1989,1991; Tatara et al. 1991), and Equation 2. The Tataramodel has been developed for a large deformation of arubber sphere compressed between two flat plates,allowing a lateral extension of the sample. We adoptthis model although the AFM tip is not quite a flatplate. By using the same symbol a as above, one of theTatara equations is given as follows by assuming that

Figure 4. (A) A collection of compressive curves obtained under the same

conditions. The abscissa is the distance from the end of compression, and

the ordinate is the compressive force. The pink, yellow, and cyan lines in

the figure are best-fitting curves based on Equation 1 (Hertz model),

Equation 2 (modified Hertz), and Equation 39 (Tatara model), respec-

tively. (B) Four possible cases of protein compression. In each figure, the

solid and dotted lines, respectively, represent relative positions of the tip,

sample, and the substrate before and after compression. Symbols corre-

spond to those defined in the text. Case 1 indicates a small deformation

from both top and bottom side of a spherical sample; case 2, a small

deformation from the top only (original Hertz model) of a mushroom

shaped sample; case 3, a large deformation from the top and bottom

(applied for the native protein) allowing lateral extension (indicated with

horizontal arrows here and in case 4); and case 4, a large deformation

from the top with lateral extension (applied for denatured protein).

Figure 5. Plot of apparent Young’s modulus of BCA II, Yapp, as a

function of IH. The abscissa is the Hertzian approach distance, IH,

defined as either IH¼ I/2 for curves 1 and 2, and IH¼ I for the rest of

the curves, where I is the experimentally observed depth of indentation

(see text for individual cases). Yapp was calculated applying Equation 2

to the experimental data. Numbers represent the following: 1, native;

2, complexed with an inhibitor; 3, in 1.0–1.5 M; 4, 2 M; 5, 3 M; and 6,

6 M GdmCl solutions.

Fig 4. and Fig 5. live 4/c

www.proteinscience.org 1451

Protein softening probed by AFM

the Young’s moduli of the tip and the substrate areinfinitely larger than that of the protein sample.

F ¼ aI3=2

H þ 3a2

2ac

� �I 2H þ 15a3

8a 2c

� �I

5=2H ð3Þ

where, ac ¼4�Y1R1

1þ �1ð Þ 3 � 2�1ð Þ :

Since a and ac are related through Y1, we can reduceEquation 3 to a single parameter equation of 39 belowby assuming �1¼ 0.40, and use the latter equation forcurve fitting to the experimental data.

F ¼ aI3=2

H þ 0:337aI 2H þ 0:0948aI

5=2H ð30Þ

The numerical coefficients of Equation 39 is onlyweakly dependent on the value of �1 between 0.35 and0.45. In the following analysis, we adopted �1¼ 0.40.The final value of Young’s modulus to be calculatedfrom a then has an uncertainty range of 65% due tothe (1� �1

2) term for the range of �1¼ 0.35 to 0.45.Equation 3 is given by Tatara (1989) as an expansionformula of the implicit relation of IH to F given below,

IH ¼ F

a

� �2=3� F

acð4Þ

where,1

ac¼ 1 þ �1ð Þ 3 � 2�1ð Þ

4�Y1R1þ 1 þ 2�2ð Þ 3 � 2�2ð Þ

4�Y2R2:

Since the Young’s modulus of silicon substrate is large,we can set ac¼ 4pY1R1/(1+�1)(3�2�1) as already given.Tatara model was applied by Liu et al. (1998) for theanalysis of compression curves of polyurethane spheres.Although both Hertz and Tatara models have been devel-oped for materials with homogeneous and isotropicphysical properties, we applied these models to nonhomo-geneous and nonisotropic protein molecules to obtain anumerical measure of rigidity of BCA II during its dena-turation process in GdmCl solutions.

In Figure 4A, experimental force curves are over-lapped with the best-fitting curves to Equation 1

(pink), Equation 2 (yellow), and Equation 39 (cyan) inthe middle part of the plot. The fitting curve based onEquation 39 reproduced the experimental data down to�60%–70% of the observed depth of compression witha constant value of Y1, which we call Yexp, supportingthe reliability of our estimate of Yexp.

The Young’s modulus, Yexp, obtained from the value ofa in each fitting curve by assuming that the Poisson’sratio¼ 0.40 andR¼ 2.3 nm (based on the estimated valuesof R1¼ 2.5 nm and R2¼ 30 nm), was 806 10 MPa forEquation 1 in the initial 10% of the total compressiondepth, and 756 10 MPa for both Equation 2 and Equa-tion 39. If we force Equation 1 to fit to 70% of the datastarting from zero compression, Yexp was increased to1506 20 MPa, suggesting that we would get approxi-mately twice as largeYexp from a forced fitting of Equation1 for a large deformation of protein molecules. Since, asnoted above, Equation 39 could be fitted nicely up to 60%–70% of the total compression depth with a single param-eter as shown in Figure 4A, we think it is the mostreliable model and results cited in Table 1 are all basedon Equation 39. Deviation of the experimental points fromEquation 39 is most likely due to nonconstant Young’smodulus of BCA II, but we need more precise data inhigher compression regions, which will be left for futurestudies. In the following analysis of compression curvesunder different conditions, we used Equations 39 and 2 toobtain the value of a and calculated Yexp, which covers60%–70% of the data range in each case. The results aresummarized in Table 1.

Experimentally, it is not likely that the protein moleculeswere always caught at the very apex of the AFM tip, andconsequently, the compressive force was applied not nor-mal to the substrate plane but with an angle � against thesurface normal. In such cases, neither the Hertz nor theTatara model can be applied without modifications, butcorrections to the theoretical results are beyond the scopeof this work. To amend this problem and apply the theo-retical models as much as possible, we collected forcecurves with largest values of the depth of compression ineach case presented in this work so that the corrections dueto nonzero �, where it is necessary, remain within10%–20% of the exact value.

Table 1. H and Yexp obtained from the best-fit parameters in the initial to midrange of compression of BCA II

using Equations 39 and 399 in different concentrations of GdmCl

0 M GdmCl 0 M GdmCl + inhibitor 1.5 M GdmCl 2.0 M GdmCl 3.0 M GdmCl 6 M GdmCl

H (nm) (height) 3.56 0.1 2.06 0.2 36 0.5 4–5 5–7 8–10

Yexp (MPa) for �=0.40 756 10a 656 10a 156 5b

256 5a4–6b 3–6b 26 0.5b

56 1a

aData were fitted to Equation 39 by setting IH= I/2, where IH is theHertzian approach distance and I is the experimentally observed depth of compression.bData were fitted to Equations 39 or 399 by setting IH= I.

1452 Protein Science, vol. 14

Afrin et al.

TheYoung’s modulus of BCA II obtained above is quitea low value compared with a previously reported valueof 500 MPa for lysozyme adsorbed on mica (Radmacheret al. 1994). Structural differences of the two proteins andthe experimental conditions (lysozyme was physicallyadsorbed instead of cross-linked to mica surface) may beresponsible for the observed difference in the Young’smodulus (see Discussion).

When the enzyme inhibitor was added to the samplesolution, the value of Yexp became slightly lower thanthat of the native enzyme but the height was muchreduced, indicating that the core of BCA II becamemore rigid than in the native state (Table 1).

The increased stability of the inhibitor (acetazolamide)-bound formof CAhas been reported (Almstedt et al. 2004).TheresultofX-raycrystallographyshowsthedistributionofthe thermal B-factor in the native and inhibitor-bound formof human CA. In the native state, not surprisingly, thesurface residues have larger B-factors compared with theinternal residues, but in the inhibitor-bound form, the dif-ference is more pronounced. For example, the results inProtein Data Bank (PDB) codes 1CAII and 1A42, respec-tively, for the native and inhibitor-bound forms show thistendency. If the B-factors can be taken as an indicator ofrelativerigidity, therigidityof the surface residues is reduced,whereas that of internal residues enhanced. Mechanicalrigidity of the inhibitor-bound form of BCA II, as cited inTable 1, shows a slightly reduced Young’s modulus in theinitial stage of compression, but the total compressiondepthis only 60% of the native protein. We temporarily inter-preted the result as indicating a softened surface residuesand incompressibly rigidified internal core structurewith theforceof�2nN.Compressionof theproteinmolecule inbothnative and inhibitor-bound formsmust be carried out, in thefuture, with an improved accuracy in a large force range.

Results in 1–1.5 M GdmCl

In 1–1.5 M GdmCl, BCA II is known to be in the‘‘molten globule’’ state (Uversky 1993; Andersson et al.2001; Bushmarina et al. 2001), and as expected, thepulling force curves under this condition showed thatthe protein yielded to much less force in the initial30-nm extension and was stretched as a flexible chainthereafter up to almost the full contour length of thechain before the covalent cross-linking system was sev-ered (Figure 6A). The result indicated BCA II lost thehighly resistant b-core structure observed under thenative condition. Reference experiments using unmodi-fied tips confirmed that the nonspecific adhesion forcewas always <0.05 nN. F–E curves revealed that themechanical resistance of the protein against the tensileforce was in the range of 0.2–0.3 nN up to 80 nm inextension, except in a few cases where there was still a

remnant of force peaks in the 30-nm region. This is aclear indication of the loss of the substructures respon-sible for the strong mechanical resistance in the nativeprotein, which was so prominent in Figure 3A.

Analysis of the approach part of the force curvesgiven in Figure 6B revealed that, under this condition,BCA II was very much softened compared with thenative state with no change or even a small decrease inH. It is most likely that the protein is somewhat flat-tened on the substrate due to the loss of rigid tertiarystructure. Equation 39 was fitted to the data in the initial60% of the data range, assuming IH¼ I to giveYexp¼ 156 5 MPa. Since the sample is small, it is prob-ably not strictly compressed from the top only. In such acase, the true value of Young’s modulus should liebetween the two extreme cases of IH¼ I and IH¼ I/2.Table 1 gives the results for both cases.

Figure 6. (A) F–E curves under 1.5 M GdmCl showing various degrees

of resistance against tensile force in the initial extension up to 40 nm,

after which chains were extended as a flexible polymer. Inset is a

collection of representative individual curves. (B) Collection of com-

pressive curves with a fitting curve based on Equation 39 as a white

line. The abscissa is the distance from the end of compression, and the

ordinate is the compressive force.

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Protein softening probed by AFM

Results in 2 and 3 M GdmCl

The pulling curves in 2 M GdmCl were similar to thoseobtained in 1.5 M GdmCl, in that they were character-ized by initial plateau region of 0.2–0.3 nN followed bya temporal drop in the force value before the final steeprise toward the maximum extension of the chain (datanot shown). In 3 M GdmCl, the tensile response of theprotein was very similar to that of the fully denaturedproteins in 6 M GdmCl in Figure 7A (data not shown).

Results in 6 M GdmCl

In the presence of 6 M GdmCl, force curves showed anearly ideal behavior for a flexible worm-like chain. Acollection of experimental pulling curves is given inFigure 7A together with a fitting curve for a worm-likechain model, as described by Bustamante et al. (1994)and Smith et al. (1996), using the formula that followswhere kB, T, p, E, and E0 are the Boltzmann constant,temperature, the persistence length of the chain, chainextension, and the length of the fully extended chain,respectively.

F ¼ kBT

p

�1

4 1� E=E0ð Þ2� 1

4þ E

E0

�ð5Þ

The fitting curve in Figure 7A was obtained byadjusting P¼ 0.2 nm and E0¼ 90 nm. The result sup-ports that the protein is fully denatured under this con-dition.

In Figure 7B, compression curves are overlapped witha fitting curve based on Equation 399 given below, whichhas different coefficients from Equation 39because of thedifferences in R1 and R.

F ¼ aI3=2

H þ 0:174aI 2H þ 0:0250aI

5=2H ð300Þ

The value of maximum compression, H (96 1 nm),obtained from the above analysis is expected to beapproximately equal to the height of the denaturedprotein on the solid surface because the data cluster ataround x¼ 0 for a compressing force of 0.6–1.5 nN.Thus H corresponds to the maximum depth of compres-sion and can be taken as the radius of denatured pro-tein. Taking R1¼ 9 nm and R2¼ 30 nm, we obtainR¼ 6.9 nm. Yexp of the denatured protein was in therange of 26 0.5 MPa (IH¼ I) (Fig. 5, curve 6). If theprotein is assumed to be compressed from both top andbottom, Yexp would be 56 1 MPa.

It has been suggested that the height of randomlycoiled polymer tethered on one end to a solid surfaceroughly corresponds to its radius. Since the unperturbedend-to-end distance of denatured BCA II is estimated tobe in the range of 14–15 nm according to the equation

given by Tanford (1968), the height probed by theAFM tip is close to one half of the end-to-end distancebut significantly larger than the Stokes radius, RS¼ 5.1–5.2 nm as reported by Uversky (1993). The radius ofgyration of randomly coiled polymer has been given asRG¼ [3/2]RS (Strobl 1997), which is equal to 7.7 nm inthis case and close to the value of H. Therefore, weconcluded that the cantilever we used in this worksensed the presence of denatured protein at a distancefrom the substrate close to either a half of the end-to-end distance, or the radius of gyration.

It should be added that the effect of the hard sub-strate on the compression experiment of small samplemust be taken into consideration in a more precise workin the future (Akhremitchev and Walker 1999).

Discussion

Unfolding and refolding of globular proteins has beenintensively studied to gain an understanding of the

Figure 7. (A) Collection of F–E curves in 6 M GdmCl fitted with the

interpolation formula (Equation 5 in text) of the WLC model in red.

Inset is a collection of representative individual curves. (B) Collection

of compressing curves under the same conditions with a fitting curve

based on Equation 399 as a white line. The abscissa is the distance from

the end of compression, and the ordinate is the compressive force.

Fig 7. live 4/c

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Afrin et al.

mechanisms of spontaneous creation of their three-dimensional conformations that have biological activity.BCA II has been regarded as one of the few examplesproviding clear evidence of the presence of intermediatestates in the transition region, different from either thenative or the fully denatured states. The intermediatestate has been characterized by a variety of optical andhydrodynamic methods, but its mechanical rigidity hasnot been studied because such a property is only mean-ingful when measured at the single molecular level. Therecent development of single molecule force spectro-scopy has enabled us to measure the mechanical proper-ties of individual molecules of proteins and otherbiological polymers. In this article, we probed the pro-gressive softening of BCA II under mild to fully dena-turing conditions by using an AFM.

Under native conditions, the pulling curve of BCA IIwas characterized by a short extension of 15–30 nm beforethe covalent system broke down. The inability of the chainto be extended beyond this level is most likely to be theresult of a strong friction between the C-terminal andN-terminal chains in the knot region. From the compres-sion curves, the molecule was shown to have a maximumcompression depth ofH� 3.56 0.1 nm, which in this caseis most likely to be less than the sample height, and byusing Tatara equation, Yexp was found to be in the rangeof 756 10MPa for�60% of the compression range fromthe onset of compression. The large difference observedbetween the Young’s modulus of BCA II and that oflysozyme as measured by Radmacher et al. (1994) is prob-ably due to, first, to the small size of the latter protein thatis structurally reinforced by four disulfide bonds and,second, to the multipoint adhesion of lysozyme to thesubstrate, whereas BCA II in this work cross-linked tothe substrate through a single cross-linker.

BCA II in the early transition region of 1.0–1.5 MGdmCl gave a similar or slightly lower value of H(�3 nm) than that of the native protein but had a muchsmaller starting value ofYexp<20MPa, implying that theprotein molecules were significantly flattened comparedwith the protein under native conditions but still not assoftened as in 6 M GdmCl. A similar conclusion can beobtained from the analysis of pulling curves obtainedunder the same condition. Thus the AFM experimentssuggested that the protein was in a different state fromeither the native or the denatured states, confirming thewell-known fact of the non–two-state transition of dena-turation of this protein induced by GdmCl. What is newin this work is the actual measurement of the F–E curves,together with the modulus of elasticity of the intermediatestates of denaturation.

It is noteworthy that the presence of softened proteinmolecules was detected in AFM experiments even in thenative condition, and that the relative population of the

softened protein molecules increased as the concentrationof GdmCl was increased. Such heterogeneity in the molec-ular population observed in pulling experiments can bepursued further in future study. As well, the extent of chainstretching at the final rupture point was short under nativeconditions for the majority of the molecules, but this lengthgradually increased as the concentration of the denaturantwas increased from 1 to 3 M, where the majority of theprotein molecules showed denatured type F–E curves. Bothof these aspects in BCA II denaturation may have complextime dependencies and are left for future investigation.

Under fully denaturing conditions, each tetheredmolecule on a solid surface is expected to form a struc-ture known in polymer physics as a ‘‘mushroom’’(Strobl 1997; Jones and Richards 1999). The height ofmushroom BCA II in 6 M GdmCl (�8–10 nm) wassignificantly greater than the Stokes radius of the sameprotein (�5 nm) under a similar denaturing condition. Ifwe take the known relationship between RS andRG¼ (3/2)RS known for an ideal Gaussian chain, weobtain RG¼ 7.5 nm for an ideal Gaussian chain, whichis closer to H than RS is. Furthermore, the end-to-enddistance of an unperturbed Gaussian chain of polypep-tide having 260–290 amino acid residues can be esti-mated as �14–15 nm according to the empiricalequation given by Tanford (1968), which is also closeto, but slightly less than, twice the experimental Hobtained in 6 M GdmCl. Although the value of H isexpected to depend on the force constant of the canti-lever and the radius of the tip, such dependence was notstudied in this work.

In summary, we have, for the first time, measured themechanical softness of a globular protein under progres-sively denaturing conditions and proved that an AFM hasthe capability to follow the denaturation process at thesingle molecular level. It is especially interesting to havefound that AFM successfully distinguished the intermedi-ate states of denaturation of BCA II from either the nativeor a fully denatured form. Changes of physical propertiesof protein molecules either adsorbed or immobilized onvarious solid surfaces are attracting the attention ofresearchers concerned with proteins at interfaces. Ourmethod will enlarge the repertoire of methods availableto study such processes at the single molecular level.

Materials and methods

Protein

Recombinantly modified BCA II with two cysteine residues eachat the N or the C terminus and a His6-tag sequence outside of theN terminus containing extra 63His tag sequence was producedas described previously and purified by the metal chelating chro-matography (Ni-NTA agarose) and affinity chromatographyprepared with p-aminomethylbenzenesulfonamide followed by

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Protein softening probed by AFM

gel filtration chromatography (Alam et al. 2002). The total con-tour length between two cysteine residues is �96 nm, assumingthat the length of one amino acid residue is 0.37 nm. BCA II hasrecently been shown to have virtually the same three-dimensionalstructure as its human counterpart (Saito et al. 2004), and themodified protein used in this work had full enzymatic activity(Wang et al. 2001). The presence of extra His-tag residues inter-fered neither with the structure nor with the activity of theenzyme, and their effect was not explicitly considered in thiswork. We also confirmed by measuring the intrinsic fluorescenceof the modified protein that it was denatured in the concentrationrange of 1–3 M of GdmCl as reported for wild-type protein(Wong and Tanford 1973; Bushmarina et al. 2001).

Chemicals

Succinimidyl 6-[39(2-pyridyldithio)-propionamido]hexanoate(LC-SPDP) was purchased from Pierce and stored underhighly dehydrated and low temperature conditions. The sila-nization reagent, 3-aminopropyltriethoxy silane (APTES), waspurchased from Shin-Etsu Chemicals. GdmCl, p-amino-methylbenzenesulfonamide, and other chemicals were pur-chased from Sigma Chemical Co. All the reagents were usedwithout further purification.

Silicon substrate and protein immobilization

Crystalline silicon wafers were purchased from Shin-EtsuChemicals, cut into �10 mm3 10 mm square pieces, andcleaned first in chloroform, then in ethanol, and finally inMilliQ water. Wafers were then treated in Piranha solution(H2SO4 :H2O2¼ 3 : 1) and washed with MilliQ water. Theywere further treated in an ozone cleaner for 2 min under O2,then for 20 min UV irradiation, and finally for 6 min under N2.Silanization with APTES was performed as described pre-viously (Afrin et al. 2003). The silanized wafers were thenderivatized with the hetero-bifunctional cross-linker LC-SPDP,which has the amino-reactive succinimidyl group and isexpected to form a covalent cross-link with amino-silanizedsilicon surface. The other reactive group of the cross-linker,pyridyldithio group, is reactive toward -SH groups on therecombinant BCA II molecules, thus the cross-linker was usedto covalently immobilize BCA II on the silanized silicon sur-face. The use of covalent cross-linkers to immobilize proteinmolecules to the substrate was an important step in this workbecause most of the AFM measurements were done in thepresence of the denaturant GdmCl, which otherwise denaturesand washes off noncovalently attached proteins on the sub-strate. The success of the immobilization of the protein on thesilicon surface was confirmed by a TRIFT III TOF SIMS(time-of-flight secondary ion mass spectroscopy) apparatus,which demonstrated (m/z) peaks at 110, 120, and 130, corre-sponding to nitrogen containing fragments of C6H10N2,C8H10N, and C9H8N, respectively (data not shown).

The flatness of the bare and APTES-modified siliconsubstrate was measured by imaging the surface roughness,which was found to be within 60.5 nm.

AFM experiments

NanoScope IIIa (Digital Instruments) was used at room tem-perature (�25�C) with the force curve mode. For force curve

measurements, a silicon substrate with immobilized BCA IImolecules was placed under 50 mM Tris-sulfate buffer (pH7.5) containing various concentrations of GdmCl between 0and 6 M. A modified tip with the cross-linker LC-SPDP wasthen brought in contact with the sample layer on the AFM.During their brief contact, covalent bonds were occasionallyformed between the remaining -SH groups on immobilizedBCA II and the dithiopyridyl groups on the tip. Covalentbond formation was verified after recording force curves thatshowed the presence of tensile material between the tip and thesubstrate and ended in a complete release of the cantilever withthe final rupture force of �1.0–2.5 nN (Afrin et al. 2003). Toavoid simultaneous formation of more than two cross-linksbetween the tip and the proteins, the probability of covalentbond formation against the total number of mutualapproaches was kept to <20% by adjusting the protein con-centration to <50 mg/mL in the immobilization step of BCAII. In compression experiments, an AFM tip was pushed ontothe sample protein with the final force exceeding 1 nN toassure the maximum compression. When the tensile character-istics of the protein were probed in the retracting regime of theforce curves, the initial compression force was kept as small aspossible to guarantee reversible compression of the protein,i.e., in the range of 0.3–0.5 nN. In addition, since experimentswere done in solutions having an ionic strength >100 mM,electrostatic contribution to the tip–sample interaction wasconsidered negligibly small in the analysis of the force curves.

The silicon nitride NPS probes used in the AFMmeasure-ments were purchased from Digital Instruments (presentlyVeeco Instruments Japan). Triangle cantilevers with a nominalforce constant of 0.06 N/m were used in this experiment. Theywere similarly functionalized with APTES and LC-SPDP asdescribed above for silicon substrates. The spring constant ofcantilevers used in the experiment was calibrated by the ther-mal vibration method (Hutter and Bechhoefer 1993). Collectedforce curves were analyzed using SPIP (Image Metrology),Microsoft Excel (Microsoft Corp.), and Igor Pro software(Wavemetrics).

Acknowledgments

This work was supported by a grants-in-aid #99R16701(Research for Future Program) and #15101004 (BasicResearch [S]) from the Japan Society for Promotion of Science(JSPS) to A.I. R.A is a recipient of JSPS postdoctoral fellow-ship. We thank Alvac Phi Co. for conducting TOF SIMS mea-surements. We thank Professor Uno Carlsson of LinkopingUniversity (Sweden) for critically reading the manuscript.

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