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Hidden variety of biotin–streptavidin/avidin local interactions revealed bysite-selective dynamic force spectroscopy
Atsushi Taninaka, Osamu Takeuchi and Hidemi Shigekawa*

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Published on 20 August 2010 on http://pubs.rsc.org | doi:10.1039/C0CP00259C

Received 18th April 2010, Accepted 14th July 2010
DOI: 10.1039/c0cp00259c
By site-selective dynamic force spectroscopy realized with the combination of cross-linkers and anatomic
force microscope with a force feedback system, we have revealed, for the ﬁrst time, that the slight
diﬀerence between the local structures of amino acid residues at the middle sites,
SER45 and THR35 for streptavidin and avidin, respectively, strongly aﬀects the microscopic reaction
processes, i.e., the variation governs the type of bond as well as the ﬁne structure of the potential
landscape. For streptavidin, a bridged or direct hydrogen bond is induced depending on the molecular
structure in the buﬀer solution. For avidin, in contrast, only a direct hydrogen bond is observed for all
the buﬀer solutions used in the experiment. Since ﬁnal functions in a system are realized through theassembly of local eﬀects, the obtained results indicate the importance of analyzing the reaction processes
with respect to the local structures of molecules, for further development of nanoscale functional devices.

1. Introduction
The speciﬁc natures of functional molecules are governed by thecharacteristics of their local structures and properties in manycases.1–4 In fact, even in the reaction dynamics of complex materials
such as proteins, a variety of local structures, for example, aﬀect thesteric hindrance of enzyme reactions;1–3 thereby, they play animportant role in governing total reaction processes. Therefore,
for further understanding of molecular recognition properties andtheir application to the development of functional devices, it is ofgreat importance to analyze the reaction processes of moleculeswith respect to a variety of local structures and their properties.
However, such an analysis has not been successfully carried out
because of the diﬃculty in discriminating the characteristics of theselected site from complex interactions.
To investigate the microscopic processes, we performed siteselective dynamic force spectroscopy (DFS)5–28 on typicalligand and receptor, namely, streptavidin/avidin–biotin
systems. Site-selective DFS is the method which enables us
to investigate the site-dependent stochastic process of a chemical
reaction of molecules at the single-molecule level.23,24
Fig. 1 shows schematics of the bonding structures of
streptavidin/avidin–biotin systems.1–3 As is shown in
Fig. 1(a), the binding pocket of streptavidin has several
reaction sites with a hydrogen-bonding network, which areclassiﬁed into the following three groups depending on the
distance from the bottom of the binding pocket: (1) innerbinding sites of amino acid residues SER27, ASN23, TYR43
and ASP128, (2) middle binding sites SER45 and THR90, and(3) outer sites ASN49 and SER88.1,2 On the other hand, as
shown by the schematic structure of the avidin molecule inFig. 1(b), avidin has three amino acid residues that are
diﬀerent from those in streptavidin.3

Institute of Applied Physics, CREST-JST, University of Tsukuba,
1-1-1 Tennodai, Tsukuba, Ibaraki, Japan.
E-mail: hidemi@ims.tsukuba.ac.jp; Web: http://dora.ims.tsukuba.ac.
jp/; Fax: +81-29-853-5276; Tel: +81-29-853-5276

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Phys. Chem. Chem. Phys., 2010, 12, 12578–12583

Fig. 1 Schematics of the bonding structures of (a) streptavidin–biotinand (b) avidin–biotin complexes. Three diﬀerent sites are indicated by
red letters. Orange dotted and blue circles indicate the inner andmiddle binding sites discussed in text.

Although notable diﬀerence in the characteristics of two
molecules for chemical reactions cannot be probed by thermodynamic method, the local variations between streptavidin andavidin may provide a diﬀerence hidden behind the averagedfunction when microscopic potential landscapes are directly
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probed. In fact, in the pioneering work7,8 by DFS on thesematerials, although details could not be clariﬁed in an
ordinary way, a slight diﬀerence in the value of potential
positions was observed between biotin/streptavidin (0.5 nm)
and biotin/avidin structures (0.3 nm). Furthermore, in aprevious paper, it was reported that reaction sites of streptavidin
and biotin form a bridged bond in 0.01 M phosphate (pH 7.4),
while they form a direct hydrogen bond in 0.05 M sodium
nitrate (pH 7),23–25 suggesting a signiﬁcant eﬀect of ion species
in buﬀer solutions on the bond formation, which may also
result in a diﬀerent bond formation for avidin. Therefore,
probing to obtain a deeper understanding of these typicalligand–receptor systems is of great importance for providing ageneral basis for designing and controlling the mechanism ofchemical reactions between two more complicated functional
molecules.
Using site-selective DFS, eﬀects of the local variation in thetype of amino acid residues in streptavidin and avidin
molecules on their reaction dynamics with a biotin molecule
were studied in detail at the single molecular level, and criticaleﬀects, which had been hidden behind the averaged function,
were revealed, for the ﬁrst time.

2. Experimental
2.1

Sample preparation and buﬀer solutions

Fig. 2(a) shows a schematic illustration of a sample preparation.
A gold-coated cantilever (Bio-Lever, Olympus, 6 pN nm1

and 30 pN nm1, rectangular shape) was immersed into asolution of 8-amino, 1-octanethiol hydrochloride molecules(1 mM in ethanol) for 48 h to form a closely packed
self-assembled monolayer (SAM) with amino groups on thesurface. After rinsing with ethanol, the cantilever was immersed
into a solution of biotin-PEG3400-COO-NHS molecules(Shearwater Polymers, 0.1 mM in ethanol) for 20 h to ﬁx a
biotin (biotin-PEG) molecule onto the probe apex. Finally, thecantilever was rinsed with ethanol.
For probing middle sites selectively, streptavidin/avidin
molecules were ﬁxed via a streptavidin–maleimide or an
avidin–maleimide structure on the substrate.23,24 First, as a
substrate, a thin gold ﬁlm (100 nm) was evaporated onto a
freshly cleaved mica surface in a high vacuum at 400 1C andﬂame-annealed using a hydrogen gas burner for 30 s. Then, thesubstrate was immersed into a solution of 1,10-decanedithiol/
1-octanethiol (1/100 ratio) (1 mM in ethanol) for 48 h to forma closely packed SAM with thiol groups on the surface.
The substrate was immersed into a solution of streptavidin–
maleimide (10 mg l1 in phosphate-buﬀered saline (PBS)(pH 7.4) : N,N-dimethylformamide = 99 : 1) for 5 h to ﬁx
streptavidin molecules to the substrate. The avidin–maleimide
was synthesized using avidin molecules (1 mg ml1 inphosphate-buﬀered saline (PBS)) and a sulfo-SMCC of 0.1 mg,
and the activated avidin was puriﬁed by desalting on a column
and an HPLC (High performance liquid chromatography).
The formation of closely packed SAMs on the probe apex andsubstrate was necessary to decrease the strong interaction
between the gold layers on both surfaces.
To avoid multiple-bonding events, as mentioned above, thedensity of the target molecules in the SAM was reduced so thatthe probability of bonding became 5–10% for each tip–sample
approach. Furthermore, only single ruptures were counted toremove the errors caused by the eﬀect of multiple-rupture
events on the analysis.
For buﬀer solutions, in addition to 0.01 M phosphate(pH 7.4) and 0.05 M sodium nitrate (pH 7), which were usedin previous studies of streptavidin,23–25 we prepared 0.05 M
carbonate (pH 10) that has a structure similar to that of
sodium nitrate but has a diﬀerent pH.
2.2 Measurement proceduresFig. 2 (a) Schematic illustrations of the apex of a modiﬁed cantilever.
Streptavidin or avidin is ﬁxed to a SAM of 1,10-decanedithiol/
1-octanethiol (1/100 ratio) mixed solution on a Au-coated substrate
via a streptavidin–maleimide or avidin–maleimide structure. (b)–(d)Procedures of obtaining potential barrier position shown in (e).

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In DFS, the unbinding force applied to a molecular pair isincreased at a constant rate, and the force required to rupture
the bond is measured. Through the analysis of the relationshipbetween the modal rupture force and the logarithm of theloading rate, microscopic potential barrier landscapes(distance from the potential minimum) and the lifetimes ofbonds can be obtained.5–28 The lifetime of a molecular bond isdependent on potential barrier height. When a tensile force isapplied to a bond, the potential landscape is deformed; thus,
barrier height decreases. For a constant loading rate of thetensile force, the probability distribution of the rupture force
can be expressed asPðf Þ ¼ C exp





ðf  f  Þxbðf  f  Þxb
exp 1  exp
ð1Þ
kB T
kB T

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where P(f), f, f*, xb, kB, T, and C are the probability distributionof the rupture force, the rupture force, the modal rupture
force, the potential barrier position, the Boltzmann constant,
the temperature and a constant, respectively.7,8,23–25 According
to eqn (1), f* linearly depends on the logarithm of the loading
rate r0((=df/dt)) as shown by

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f ¼

kB T
xb


ln r0 þ lntoffð0Þ xb
kB T

ð2Þ

where toﬀ(0) is the lifetime of a molecular bond. Eqn (2)indicates that the potential barrier position can be obtained
from the slope of the linear relationship.5–28
When using eqn (1) or (2), it is essential to maintain aconstant loading rate.13,14,23–27 When the DFS measurement iscarried out by an atomic force microscope (AFM) with across-linker molecule, however, a constant retraction velocity
does not result in a constant loading rate because of thestretching of the cross-linker molecule. Namely, controllingof the applied force to maintain a constant loading rate is ofgreat importance. An AFM system with a feedback loop wasused to satisfy this requirement,23–27 which enables the ﬁnecontrol of low loading rates to reduce the eﬀect of the softcross-linker that connects a sample molecule to the tip.23–27
The AFM system also enables us to realize a high sampling
rate (100 kHz) to obtain a suﬃcient amount of data at a highloading rate.23–25 Furthermore, since AFM measurement isstable under various buﬀer solutions, diﬀerent buﬀer solutions
with diﬀerent pH values can be used.
For a selected loading rate, many rupture forces aremeasured from force curves (Fig. 2(b)). The measured rupture
forces are shown in a histogram, from which the modal
rupture force is obtained for each loading rate measurementby theoretical ﬁtting (Fig. 2(c)). Then, the modal rupture forceis plotted as a function of the logarithm of the loading rate
(Fig. 2(d)): therefore, information concerning the energy
landscape of the interaction is derived from the relationshipbetween the modal rupture force obtained from the histogram
and the loading rate of the unbinding force. In addition, thewidth of the histogram also provides the potential barrier
position by analysis using eqn (1), which can be used to
examine the validity of the results obtained from the slope
shown in Fig. 2(d).
For our experiment, the loading rate was controlled between
10 and 105 pN s1, and 5000–10 000 approach and retract
cycles were carried out to form a histogram for each loading
rate measurement.

3. Result and discussion
Fig. 3(a) and (b) show two diﬀerent forms of a typical force
curve obtained using the system that we developed23–25 asfunctions of (a) distance and (b) time. The distance represents
the length of the cantilever retraction after zero force wasmeasured. Since the cantilever was moved in until the force
reached 50 pN for setup, there exists a negative region for the
distance. Although the force does not show a linear change in(a) owing to the existence of a PEG molecule, a linear change
is shown in (b). This result also indicates that a constant
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Phys. Chem. Chem. Phys., 2010, 12, 12578–12583

Fig. 3 Two diﬀerent forms of a typical force curve obtained using thesystem that we developed23–25 as functions of (a) distance and (b) time.

loading rate is achieved using the AFM system that wedeveloped.
When a molecular cross-linker is used to ﬁx a sample to atip, the condition of a constant loading rate cannot besatisﬁed. By considering the eﬀect, several models have been
proposed.13–22 However, since the AFM system with a feedback loop that we used satisﬁes the condition of a constantloading rate, the eﬀect of the soft cross-linker that connects asample molecule to the tip can be satisfactorily minimized.
Therefore, eqn (1) and (2) of the Bell–Evans model can be usedwithout any modiﬁcations for the analysis of the obtained
results.21–25
As described in the Experimental section, for a selectedloading rate, many rupture forces were measured from force
curves. The rupture forces were summarized in a histogram,from which the modal rupture force was obtained for eachloading rate measurement.
Fig. 4 represents typical histograms of the rupture forces
obtained for the streptavidin–maleimide in (a) a pH 7.4
phosphate buﬀer solution at the loading rate of 1115 pN s1,(b) a pH 7 sodium nitrate solution at the loading rates of1447 pN s1 and (c) a pH 10 carbonate buﬀer solution at theloading rates of 1382 pN s1, and for the avidin–maleimide in(d) a pH 7.4 phosphate buﬀer solution at the loading rate of558 pN s1, (e) a pH 7 sodium nitrate solution at the loading
rates of 678 pN s1 and (f) a pH 10 carbonate buﬀer solution
at the loading rates of 680 pN s1. The modal rupture forceswere obtained by ﬁtting using eqn (1) based on the DFS
method.
Fig. 5 shows the relationships between the modal rupture
force and the logarithm of the loading rate obtained in 0.01 M
phosphate buﬀer solution (pH 7.4), 0.05 M sodium nitratesolution (pH 7) and 0.05 M carbonate buﬀer solution (pH 10).
For streptavidin (Fig. 5(a)), formations of a large potential
barrier position (0.68  0.01 nm) in the phosphate buﬀer solution
and a short (0.26  0.01 nm) potential position in the sodium
nitrate solution, which were observed in the previous study,23–25
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Fig. 4 Typical histograms of the rupture forces obtained for the
streptavidin–maleimide and avidin–maleimide structures in phosphate,sodium nitrate and carbonate buﬀer solutions at certain loading rates.
The solid green lines show the theoretical ﬁtting curves obtained usingeqn (1), from which the modal rupture forces are obtained. In addition
to the analysis of the slope of Fig. 5, potential barrier positions xb can be
estimated by analyzing the shape of the rupture force distribution.21–25
The potential barrier positions xb obtained from each distribution in
Fig. 4, using the method discussed in ref. 23–25, are shown in theﬁgures.

have been conﬁrmed again. In addition, a similar short potential
barrier position (0.24  0.01 nm) was observed also in the
carbonate buﬀer solution.
Here, since the distance between the inner and outer sites isabout 1 nm, the bonds with a potential barrier position larger
than 1 nm (indicated by the slopes of 1.5  0.2 nm and1.8  1.2 nm in Fig. 5(a) and (b), respectively) are not clear.
In the pioneering work by Merkel et al.7 using phosphatebuﬀer solution, they observed a potential barrier longer than
1 nm (B3 nm) only for avidin.7 In our experiment, we also
observed this long potential barrier position only for avidin forphosphate buﬀer solution, which is in good agreement with
Merkel et al.’s results.7
On the other hand, the long potential barrier positions werecommonly observed for streptavidin and avidin in nitrate andcarbonate buﬀer solutions. However, since the distance
between the inner and outer reaction sites is about 1 nm,
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Fig. 5 Relationship between the modal rupture force and thelogarithm of the loading rate obtained for (a) streptavidin–biotinand (b) avidin–biotin complexes. Colored lines are slopes for indication,and the potential positions shown together are those obtained from theslopes. The lines with two components in (a) and (b) represent theresults of ﬁtting all the data including those obtained in nitrate andcarbonate buﬀer solutions, respectively. The potential barrier
positions at middle reaction sites obtained from the data for eachsolution are summarized in Table 1.

further study is needed to clarify the mechanism of theinteractions to provide the large potential barrier positions.
We leave this issue for future study.
The potential barrier position of 0.68  0.01 nm formed in
the phosphate solution is close to the value obtained in 0.05 M
ammonium sulfate solution for streptavidin (Fig. 6). Similar to
the results obtained in a phosphate solution, only one potential
barrier position, estimated at 0.66  0.03 nm, was observed,
which was conﬁrmed by X-ray diﬀraction method to be abridged bond formed by SO42 between biotin and amino acidresidues SER45 and SER27 of the streptavidin molecule.2
These results support the fact that the streptavidin–biotinbond made in a phosphate buﬀer solution is a bridge bondformed by phosphate buﬀer molecules.
On the other hand, the barrier positions observed in thesodium nitrate and carbonate buﬀer solutions (see Table 1) areconsistent with that obtained by MD calculation for the
direct bond between SER45 and the ureido oxygen of biotin(0.26 nm).28 In general, a protein causes denaturation throughPhys. Chem. Chem. Phys., 2010, 12, 12578–12583

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Fig. 6 Relationship between the modal rupture force and thelogarithm of the loading rate obtained for streptavidin–biotin in a
0.05 M ammonium sulfate solution (pH 5).

the change in pH and ionic strength. Therefore, it may beconsidered that the binding potential is changed by denaturation.
However, as the protonation due to the change in pH wasobserved at TYR432 in a previous study, the reaction sites that
may change the potential barrier are the inner binding sites of
the amino acid residues such as TYR43, ASN23, ASP128 andSER27, which are not probed here because of the ﬁxed
condition of the streptavidin and avidin molecules preparedwith maleimide molecules used in this experiment.13 In fact,
the observed values of the potential positions were independentof pH, supporting the results obtained in this study.
These results indicate that, for streptavidin, both bridged
and direct hydrogen bonds can be formed at the middle
binding sites depending on the buﬀer solution, i.e., bridged
bonds (via phosphate molecules) in a phosphate solution
and direct hydrogen bonds in other (nitrate or carbonate)solutions, respectively.
For avidin, in contrast to the reaction processes observed
for streptavidin, completely diﬀerent results were obtained,that is, although the direct hydrogen bond of 0.12 nm,7–9

Table 1 Potential barrier positions in potential landscapes for theformation of the bonds of a biotin molecule with the middle bondingsites of streptavidin and avidin in various buﬀer solutions

Streptavidin

Avidin12582

pH

Fix type

Buﬀer molecule

Potential barrier
position/nm

7
10
5
7.4
7.4
6.8
7
10
7.4
6.8

Maleimide

NitrateCarbonate
SulfatePhosphate
Phosphate
Phosphate
NitrateCarbonate
Phosphate
Phosphate

0.26  0.01
0.24  0.01
0.66  0.03
0.68  0.01
0.63  0.03
0.5
0.32  0.01
0.35  0.03
0.33  0.01
0.3

PEG23,24
PEG7
Maleimide
PEG7

Phys. Chem. Chem. Phys., 2010, 12, 12578–12583

which is formed at the inner sites and should not be probed in
the experimental condition here, disappeared as expected, thebarrier position showed no marked change in any of the threesolutions. The potential barriers at 0.32  0.01 nm in a sodium
nitrate solution, 0.35  0.03 nm in a carbonate solution and0.33  0.01 nm in a phosphate solution (see Table 1) are close
to each other, the values of which are close to the result
obtained in a previous study, 0.3 nm in a phosphate
solution.7,8The potential barrier position xb can also be estimated byanalyzing the shape of rupture force distribution.21–25
The solid green lines in Fig. 4 show theoretical ﬁtting curves
obtained using eqn (1), and the potential barrier positions
xb obtained from each distribution in Fig. 4 are (a) 0.68  0.03 nm,(b) 0.30  0.02 nm, (c) 0.41  0.01 nm, (d) 0.34  0.02 nm,(e) 0.30  0.02 nm and (f) 0.33  0.02 nm. These results are
consistent with the barrier positions obtained from the slope of
Fig. 5; the values are close to 0.7 nm for streptavidin in
phosphate buﬀer solution and to 0.3 nm in other cases.
The results obtained for the bonding at the middle sites are
summarized in Table 1, where the data for each solution in
Fig. 5 were analyzed separately. For comparison, the results
reported in ref. 7, 23 and 24 are also shown.
What causes the observed variation in the bonding
processes? First, the origin of the variation between molecularbridging and direct hydrogen bonding observed for streptavidin
is considered to be the structure of the ion species in thesolutions. Since phosphate and sulfate have a tetrahedral
structure, the mechanism may be related to the structure of
the buﬀer molecules, i.e., when buﬀer molecules have a tetrahedral
structure consisting of four oxygen atoms, a molecularbridging is induced between SER45 and the biotin molecule,2
and a direct hydrogen bonding is induced between SER45 andthe biotin molecule for a buﬀer molecule with a trigonal
structure. Further experiment is in progress.
On the other hand, the diﬀerence in the type of amino acidresidues in the middle sites of the two molecules, namely,
SER45 and THR35 (Fig. 1), produces the diﬀerence observed
for streptavidin and avidin. Since the values observed foravidin are similar and independent of the type of buﬀer
solution used, these values are attributed to those of thepotential barriers formed by direct hydrogen bond between
the biotin molecule and the middle reaction sites of avidin,
such as THR35. Note that the barrier positions of the directhydrogen bonds observed for streptavidin are shorter thanthose for avidin, which probably originates from the diﬀerencein the type of amino acid residues. A theoretical analysis
including ﬁner structures of amino residues and buﬀer
molecules is necessary.

4. Conclusions
By site-selective DFS analysis,23–25 eﬀects of the local
variation in the type of amino acid residues in streptavidin
and avidin molecules on their reaction dynamics with a biotin
molecule were studied at the single molecule level, and criticaleﬀects, which had been hidden behind the averaged function,
have been revealed, for the ﬁrst time. The slight diﬀerence
between the local structures of amino acid residues at theThis journal is c the Owner Societies 2010

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middle sites, SER45 and THR35 for streptavidin and avidin,
respectively, strongly aﬀects the microscopic reaction
processes, i.e., the variation governs the type of bond as wellas the ﬁne structure of the potential landscape. For streptavidin,a bridged or direct hydrogen bond is induced depending on themolecular structure in the buﬀer solution. For avidin, in
contrast, only a direct hydrogen bond is observed for all the
buﬀer solutions used in the experiment. Since ﬁnal functions ina system are realized through the assembly of local eﬀects, theobtained results indicate the importance of analyzing thereaction processes with respect to the local structures ofmolecules, for further development of nanoscale functional
devices.

Acknowledgements
This work was supported in part by a Grant-in-Aid for scientiﬁc
research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (Young Scientists (B)).

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