Applied Physics Express 2 (2009) 085002

Site-Selective Anatomy of Step-by-Step Reactions in Ligand–Receptor
Bonding Processes Using Dynamic Force Spectroscopy
Atsushi Taninaka, Osamu Takeuchi, and Hidemi Shigekawa
Institute of Applied Physics, CREST-JST, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan
Received June 17, 2009; accepted July 2, 2009; published online July 24, 2009
We demonstrate a methodology that realizes the site-selective anatomy of molecular interactions at the single-molecule level. With thecombination of cross-linkers and the atomic force microscope that we developed to enable a precise analysis by dynamic force
spectroscopy, direct and bridged interactions at each reaction site in a typical ligand–receptor system, sreptavidin–biotin complex, wereclearly distinguished and individually analyzed for the first time, providing a greater understanding of step-by-step progress of the bonding
process. The bridging molecule at the middle reaction sites, which was assumed to be the water molecule in the previous study, wasidentified as the phosphate molecule. # 2009 The Japan Society of Applied Physics
DOI: 10.1143/APEX.2.085002

he variety and selectivity of interactions between apair of functional molecules, for example, DNA,ligand–receptor and antigen–antibody systems, playessential roles in biological processes and molecular devices
based on molecular recognition properties. As is the casefor a protein molecule, however, functions often originate
from structurally complex processes. Furthermore, suchinteractions include complicated many-body eﬀects arising,for example, from solvent parameters and the manifold
structures of functional molecules, which prevent the designof detailed functions in predetermined structures.1,2) Therefore, probing the step-by-step bonding processes induced by
the individual interactions in a molecular complex and theirvariation with the surrounding conditions is a key factor forenabling further advances in biophysics and chemistry andtheir applications.
The sreptavidin–biotin complex is a typical ligand–receptor system and has been extensively studied.3–8) Since
this system has a complicated structure when considering its
chemical reactions, a variety of processes are expected tooccur in this system depending on the operating conditions.
Therefore, probing to obtain a deeper understanding of
the energy landscapes of individual interaction sites of thistypical system is not only important in itself, but will also
provide a foundation for designing and controlling themechanism of chemical reactions between two complicatedfunctional molecules. Dynamic force spectroscopy (DFS) isa technique that enables us to study interaction between
molecules at the single-molecule level.3–8) In a pioneering
study on the DFS of the streptavidin–biotin complex, apotential landscape with two barrier widths (0.12 and0.5 nm) was observed, which was assumed to be due todirect hydrogen bonding and an indirect interaction via
bridged water molecules.3) However, the results obtained
from previous experiments are not comprehensively understood, and some controversies remain in explaining themechanism of the bond structures.8–14) In addition, basic
analysis methods for DFS are still being developed.8–14)
Therefore, a more detailed study is necessary to achieve
further advances.
In this paper, we present a new methodology realizing thesite-selective anatomy of molecular interactions based onDFS. Direct and bridging interactions at each reaction site in
the streptavidin–biotin complex were clearly distinguishedTE-mail address: hidemi@ims.tsukuba.ac.jp

and individually analyzed for the ﬁrst time using an atomicforce microscopy (AFM) system that we have developed,
providing a greater understanding of the step-by-step
progress of the bonding process.
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. When the DFS measurement is carriedout by AFM with a cross-linker molecule, a constantretraction velocity does not result in a constant loading rate
because of the stretching of the cross-linker molecule. We
have developed an AFM system with a feedback loop, whichenables the ﬁne control of low loading rates to reduce theeﬀect of the soft cross-linker that connects a sample moleculeto the tip or substrate.8–10,12) In addition, the AFM systemenables us to realize a high sampling rate to obtain a suﬃcientamount of data at a high loading rate. Furthermore, since
AFM measurement is stable under various pHs, diﬀerent
buﬀer solutions with diﬀerent pH values can be used.
To perform the site-selective analysis, we prepared two
types of cross-linker for controlling the depth of the active
reaction sites by changing the eﬀect of the geometry of thesystem on the bond formation site. Namely, streptavidin was
ﬁxed to a self-assembled monolayer (SAM) of (a) 8-amino,
1-octanethiol molecules on a Au-coated substrate via a
biotin–PEG–COO–NHS molecule (ﬂexible condition) or (b)1,10-decanedithiol/1-octanethiol (1/100 ratio) mixed solution on a Au-coated substrate via a streptavidin-maleimide
structure (modiﬁed condition). For both cases, a closely
packed SAM with amino groups was formed on a goldcoated cantilever and a biotin (biotin-PEG) molecule wasﬁxed onto the probe apex. As shown in Fig. 1, the biotin
molecule attached to the cantilever can enter deep into thebinding pocket and conjugate even with the inner amino acidresidues in streptavidin under the ﬂexible condition (a). In
contrast, under the modiﬁed condition (b), this process isprevented owing to a lack of ﬂexibility, and bonding only atthe middle and outer sites is achieved. Since the SAM
formed on the substrate has a hydrophobic characteristic themodiﬁed condition may increase the eﬀect of the hydrophobic feature of the substrate surface on the formation ofhydrogen bond. In this study, this method of site-selective
analysis is demonstrated to be eﬀective.
To avoid multiple-bonding events, as mentioned above,the density of the target molecules in the SAM was reduced
so that the probability of bonding became 5 –10% for each
tip–sample approach. Furthermore, only single ruptures were085002-1# 2009 The Japan Society of Applied Physics

Appl. Phys. Express 2 (2009) 085002(a)

A. Taninaka et al.

(b)

(a)

Fig. 1. Schematic illustration of site-selective analysis using twotypes of cross-linker: (a) flexible condition and (b) modified condition.
The distances between streptavidin and substrate are 30 nm for theflexible condition and 1:5 nm for the modified condition, respectively.

(b)counted to remove the errors caused by the eﬀect ofmultiple-rupture events on the analysis. The loading rate wascontrolled between 10 and 105 pN/s, and 5000 –10000
approach and retract cycles were carried out to form ahistogram for each loading-rate measurement. Information
concerning the energy landscape of the interaction wasderived from the relationship between the modal rupture
force obtained from the histogram and the loading rate of theunbinding force.
Figure 2(a) shows the relationships between the modal
rupture force and the logarithm of the loading rate obtainedin a 0.01 M phosphate buﬀered solution (PBS, pH 7.4) forthe (a) ﬂexible condition and (b) modiﬁed condition. The
inset shows a typical histogram of the rupture forces
obtained for the ﬁxed condition in a pH 7.4 phosphatebuﬀered solution at the loading rate of 2:95  102 pN/s,
where the modal rupture force is 31.0 pN. For the ﬂexible
condition, the gradient of the slope exhibits a marked
increase at 2  103 pN/s. In accordance with the DFS
theory,8) each slope corresponds to an unbinding process
related to one potential barrier, and the distance of thebarrier position from the potential bottom, xb , can be
deduced from the reciprocal of the gradient.3–14) Thepotential barrier positions were estimated from the twoslopes to be 0:13  0:01 and 0:63  0:09 nm, which areconsistent with the values of 0.12 and 0.5 nm obtained in aprevious study.3)
In contrast, under the modiﬁed condition, only one slope
was observed, and the potential barrier position estimatedfrom this slope was 0:68  0:05 nm. To conﬁrm the validityof this result, we plotted the values from 23 histograms
obtained using three cantilevers with diﬀerent spring
constants and shapes (6 pN/nm, rectangular, Au-coated;
30 pN/nm, rectangular, Au-coated; and 20 pN/nm, triangular, noncoated Si2 N3 ). No marked dependence on the type of
cantilever was observed.
As proposed in a recent study, the potential barrier
position can be estimated by analysis of the shape of therupture force distribution.8) The solid green line in the inset
shows a theoretical ﬁtting curve, and the potential barrier
position xb obtained from the distribution based on theFig. 2. (a) Relationship between the modal rupture force and thelogarithm of the loading rate. Results are obtained for the flexible[(i) red] and modified [(ii) blue] conditions using 0.01 M phosphate(pH 7.4) solution ( : 6 pN/nm, rectangular, : Au-coated, 30 pN/nm,rectangular, Au-coated, and : 20 pN/nm, triangular, noncoated
Si2 N3 ). The inset shows a typical histogram obtained for modified
condition in PBS (pH 7.4) at loading rate of 2:95  102 pN/s. Solidgreen line represents a theoretical fitting curve based on the methodof analysis proposed in ref. 8. The measurement of barrier position
is mainly affected by error of the spring constant of the cantilever (10 – 20%). (b) Similar experimental result obtained in 0.05 M
sodium nitrate buffer solution (pH 7) under modified condition (orangelines). The result obtained for the 0.01 M phosphate (pH 7.4) solution
shown in (a) is drawn together for comparison (blue dashed line).

method in ref. 8 is at 0.69 nm. The result is consistent withthe barrier position of 0.68 nm obtained from the slope ofFig. 2(a) and validates the measurements.
From the observation that the potential barrier of 0.13 nm
was obtained only under the ﬂexible condition and theprediction by molecular dynamics (MD) calculation, in
which hydrogen bonding with a rupture distance shorter
than 0.2 nm is attributed to the bonds formed at an innersite,15,16) the barrier position of 0.13 nm is related tothe direct bonding of inner amino acid residues such as
ASP128, TYR43, and ASN23 with the biotin molecule[Fig. 3(c)].17,18) On the other hand, from the results of an
MD calculation, a potential barrier with a distance greaterthan 0.4 nm cannot exist without a salt molecular bridge forthe case of phosphate.15,16) This suggests that the bondrelated to the potential barrier positions of 0.68 and 0.63 nm
is formed by molecular bridging between the amino acidresidues at the middle reaction sites and the biotin molecule.
To analyze the origin of the potential barriers of 0.68 and0.63 nm in more detail, we changed the buﬀer solution from0.01 M phosphate (pH 7.4) to 0.05 M sodium nitrate (pH 7),
and measurements were performed under the modiﬁedcondition. As shown in Fig. 2(b), the slope changed at aloading rate of about 102 –103 pN/s, and the two potential

085002-2# 2009 The Japan Society of Applied Physics

Appl. Phys. Express 2 (2009) 085002
(a)

(c)

(b)

(d)

A. Taninaka et al.

Fig. 3. Step-by-step progress of bonding. Bonds represented bythick blue dots are those determined in this study.

barrier positions were estimated to be 0:26  0:03 and1:6  0:75 nm. In addition, from the results of an MD
calculation,16) the direct bond between SER45 and the biotin
molecule has a potential barrier position of 0.26 nm.
Therefore, the bond related to the potential barrier positionof 0:26  0:03 nm is attributed to the potential barriers
formed by direct bonding between the biotin molecule andthe middle reaction sites of streptavidin such as SER45.
Since the bond of the potential barriers of 0.68 and 0.63 nm
does not exist in the 0.05 M nitrate solutions, the bond isformed by the phosphate molecules in the buﬀer solution,
instead of the water molecules, in contrast to the mechanism
predicted in a previous study.3) In fact, no rupture eventswere observed in puriﬁed water.
These results indicate that formation of hydrogen bond at
the inner site was inhibited under the modiﬁed condition, asexpected. As a result, bonding at the inner sites or middle
sites were distinguished and separately analyzed using theﬂexible and modiﬁed conditions. The potential barrier forthe direct bond at SER45 is consistent with the theoreticalprediction. Since nitrate molecules do not induce a bridgebonding, a slight change in the molecular structure of thesolvent may aﬀect the bonding interaction. The origin for thebond with a large barrier position (NaNO3 : 1:6  0:75 nm) isnot clear. Further experiments on these issues are now inprogress.
The lifetime of bonds can be estimated from the intercept
obtained by extrapolating the linear relationship between themodal rupture force and the logarithm of loading rate,8) asshown in Fig. 2.3–14) The rupture forces obtained for theﬂexible condition were larger than those for the modiﬁed
condition at all loading rates, as shown in Fig. 2, and thelifetimes obtained for the ﬂexible and modiﬁed conditions
were 6.4 and 1.0 s, respectively. From the result of theﬂexible condition (6.4 s), the lifetime for streptavidin–biotin–
PEG is estimated to be 12.8 s.5) This value is ten times larger
than that of the modiﬁed condition. This is caused by the factthat the direct bonds with the inner amino acid residues such
as ASP128, TYR43, and ASN23 are stable under the ﬂexible
condition because the biotin molecule attached to thecantilever can enter deep into the binding pocket, or theeﬀect of hydrophobic interaction from the substrate is lessthan that under the modiﬁed condition. From the experi-mental results, lifetime clearly depends on the type of bond(direct or bridged) and the formation site rather than the typeof buﬀer. At the middle sites, the lifetime of the direct bondwas shorter than that of the bridged bond, which was also
directly observed for the ﬁrst time.
The observed results enable us to discuss the step-by-step
progress of the bonding process: (1) A biotin molecule istrapped at the middle sites via the bridging of buﬀer
molecules since the bridge is the longest bond. (2) The
structure of the indirect interaction remains (in the case ofphosphate solution) or direct hydrogen bonding occurs(other solutions), depending on the relative strength of theinteraction [Figs. 3(a) and 3(b)]. (3) The bonding of theinner sites with the ureido oxygen of the biotin moleculeoccurs. (4) Bonding occurs between the outer sites and theend group of the biotin molecule (carboxyl), as illustrated inFigs. 3(c) and 3(d).
In conclusion, we demonstrated a methodology thatenables the site-selective anatomy of molecular interactionsat the single-molecule level. Using the combination of crosslinkers and an atomic force microscope that we developed
to enable precise analysis by DFS, direct and bridging
interactions at each reaction site in a ligand–receptor systemwere clearly distinguished and individually analyzed for theﬁrst time, providing a greater understanding of the step-bystep progress of the bonding process. This methodology will
provide a foundation for further advances in biophysics andchemistry and their applications, such as designing andcontrolling the mechanism of chemical reactions between
functional molecules.
Acknowledgment This work was supported in part by a Grant-in-Aidfor Scientiﬁc Research from the Ministry of Education, Culture, Sports,Science, and Technology of Japan (Yong Scientists B). We thank Ms. Rie
Yamashita, for her help in preparing this paper.

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085002-3# 2009 The Japan Society of Applied Physics