Small molecule – ligand interactions
Application Note NT-SE-001
Detection of binding-induced conformational changes by SAW
Oliver Vosyka, Matthias Molnar, Amit J. Gupta
NanoTemper Technologies GmbH, Munich, Germany
Abstract
Surface Acoustic Wave (SAW) biosensors
allow assessment of protein-small molecule
interactions with high sensitivity. The bipartite
signal readout of changes in phase and
amplitude of the acoustic mechanical wave
allows separate investigation of mass binding
and binding-induced conformational changes.
Here we use the well-established interaction of
maltose to its cognate receptor maltose
binding protein (MBP) from E. coli. Binding of
maltose to immobilized MBP results in a
quantifiable signal response, not only for a
change in mass, but also for the marked
conformational transition between the two
structural MBP domains. Using appropriate
controls we not only confirm the accessibility
of binding induced conformational changes
with SAW, but also amplitude-based Kd
extraction.
The E. coli maltose binding protein (MBP) is a
monomeric, ~41 kDa periplasmic protein, that is
often used as a fusion protein to either mediate
solubility of its fusion partners or as an affinity-tag
for amylose affinity purification (Raran-Kurussi and
Waugh, 2012).
Introduction
Surface Acoustic Wave (SAW) technology utilizes
mechanical waves propagating along the surface
of a piezoelectric crystal. Changes in the phase
and amplitude of the acoustic waves are
quantified and report mass binding and rigidity
changes of immobilized molecules. SAW
technology has a broad biomolecular application
range, from small molecules to membrane
samples and can measure binding kinetics and
affinities. Using acoustic signals, SAW is in many
cases superior to optical techniques as it is not
sensitive towards refraction-index changes. Thus,
SAW can assess interactions in turbid samples or
in the presence of high amounts of organic
solvents. Especially the high sensitivity that results
from measuring not only mass changes, but also
changes in conformational rigidity, renders SAW
an ideal tool to investigate the mechanism of
protein-small molecule interactions.
Figure 1. Schematic representation of conformational change
in MBP (~41 kDa) upon maltose binding (PDB 1OMP and
1ANF, Quiocho, Spurlino, & Rodseth, 1997; Sharff, Rodseth,
Spurlino, & Quiocho, 1992) (A) and in Lysozyme (~14 kDa)
upon binding of (GlcNAc)3 (PDB 1LZA and 1LZB, Maenaka et
al., 1995). Bound states are shown in blue, unbound states in
cyan. Sugar moieties colored in red. Structural alignment and
rendering was performed with Chimera.
In its cellular function, MBP is the corresponding
receptor molecule to the bacterial maltose ABC
transporter system. Upon traversing the bacterial
outer membrane, by means of a specified channel
protein, maltose is efficiently captured by MBP
and delivered to the inner-membrane ABC
transporter system for nutrient uptake (Nikaido,
1994). Structurally, MBP consists of βαβsecondary structure elements, forming two
globular domains that are discontinuous in
sequence, with the maltose binding site located in
a cleft between the two domains (Spurlino et al.,
1991). Interestingly, maltose binding to MBP
results in a marked conformational change. In a
clamp like motion, the MBP N- and C-domain
close around the associated maltose molecule
(Fig. 1A). Maltose binding occurs with an affinity of
~1200 nM,
as
previously determined
by
fluorescence quenching of intrinsic tryptophan
moieties (Telmer and Shilton, 2003).
Here we analyze the affinity of maltose to MBP,
based on mass- and conformational changes
quantified by Surface Acoustic Wave technology.
Results
immobilized ligand increases the velocity of the
surface acoustic wave and thereby diminishes the
phase lag. Despite their somewhat more intricate
nature, negative phase changes can be quantified
and analyzed. The increased structural rigidity of
MBP by the marked conformational change also
resulted in clear surface acoustic wave amplitude
changes (Fig. 2B). An increase in amplitude can
be attributed to an increase in rigidity of the
immobilized ligand, whereas a decrease in
amplitude can be attributed to an increased
flexibility.
As expected, lysozyme did not show a response,
neither in phase nor in amplitude, upon maltose
injection (Fig. 2A and B insets). As a further
control, we injected increasing concentrations of
glucose during the same run into all channels
(data not shown). Even at glucose concentrations
as high as 500 µM we could not detect a
significant response for MBP or lysozyme,
indicating that glucose, despite comparable
viscosity to maltose, did not induce artifactual
response signals, further validating the specificity
of maltose-binding induced signal responses
detected for immobilized MBP.
The immobilization of MBP on COOH-SAM chips
by means of NHS-ester bond formation was
efficient
with
22 deg
immobilized
MBP.
Unliganded COOH groups on the chip surface
were subsequently saturated by injection of
ethanolamine. As a control, we immobilized
lysozyme in a neighboring channel. Lysozyme
binds and hydrolyses sugar derivatives of bacterial
cell walls. In contrast to MBP, Lysozyme does not
undergo a conformational change upon binding to
its substrates, such as Triacetylchitotriose
(GlcNAc)3, which is used here. Thus lysozyme is
ideally suited as a control to validate
conformational change induced signal responses
of MBP (Fig. 1B). In addition, lysozyme does not
bind to maltose, and MBP does not bind to
(GlcNAc)3, so that the two proteins can be used
vice versa as a control for unspecific binding. In
addition, we used a blank reference channel to
control for potential unspecific effects on the chip
surface caused by injection of sugars.
The injection of increasing concentrations of
maltose resulted in concentration-dependent
changes in phase as well as amplitude in the MBP
channel. Binding of ~340 Da maltose molecules to
~41 kDa MBP was efficiently resolved by phase
changes of the surface acoustic wave, connected
to the increase in mass upon binding (Fig. 2A).
Note that the phase shift signal has a negative
sign. Negative phase shifts are the result of large
conformational changes occurring in addition to
mere mass binding, as a higher rigidity of the
Figure 2: (A) Phase and (B) amplitude response of MBP upon
injection of increasing concentrations of maltose (concentration
increases from blue to red). The insets show the signal
response for MBP (black) and lysozyme (grey) to injection of
500 µM maltose in comparison. The blank reference channel
was subtracted.
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The injection of increasing concentrations of
(GlcNAc)3 resulted in phase response signals for
lysozyme but not for MBP. As expected from the
crystal structures shown in Figure 1, the amplitude
response of lysozyme was negligible, as lysozyme
does not undergo a structural transition upon
(GlcNAc)3 binding. Correspondingly, the phase
signal for (GlcNAc)3 binding to lysozyme is
unbiased by the surface acoustic wave amplitude.
Figure 4: Equilibrium response signal was averaged for times
90 – 210 seconds after start of injection and plotted against
maltose concentration. The resulting dose response curve was
fitted with a simple one-site binding model to extract Kd values.
Conclusions
Figure 3: (A) Phase and (B) amplitude response of Lysozyme
upon injection of increasing concentrations of (GlcNAc)3
(concentration increases from blue to red). The insets show
the signal response for MBP (black) and lysozyme (grey) to
injection of 500 µM (GlcNAc)3 in comparison. The blank
reference channel was subtracted.
For maltose binding to MBP, the analysis of the
concentration dependence of phase and
amplitude signals in equilibrium (~200 seconds
after injection), resulted in dissociation constants
(Kd) of ~2100 nM and ~2300 nM respectively
(Fig. 4). These values are in good agreement to
published
data,
based
on
fluorescence
spectroscopy (~1200 nM, Telmer & Shilton, 2003).
The affinity determined for (GlcNAc)3 binding to
lysozyme was with ~11 µM also in good
agreement to previously published data (~13 µM,
Kumagai et al., 1992, data not shown).
Here we analyzed the binding of maltose to the
E. coli maltose binding protein using Surface
Acoustic Wave technology. We were able to
resolve the structural transition occurring in MBP
upon maltose binding, using lysozyme as a
negative control, which does not undergo
conformational changes upon binding of its
substrate. We further determined accurate
dissociation constants, not only by analysis of the
mass-binding induced phase-signal, but also by
quantification of the conformational-change
induced amplitude response. So far, highly
sensitive fluorescence detection was required to
quantify conformational changes on such a small
scale. Now, Surface Acoustic Wave sensors allow
monitoring of intramolecular conformational
transitions with impressive signal to noise ratios
for immobilized molecules. Therefore, SAW
presents an ideal tool to investigate mechanisms
of protein small-molecule interactions by
separating
mass
binding
and
resulting
conformational changes.
Materials and Methods
Protein immobilization
The target proteins MBP and Lysozyme were
immobilized at concentrations of 100 µg/mL in
immobilization buffer (10 mM sodium acetate pH
4.5 or pH 5.5 respectively) on a COOH-SAM
surface using PBS pH 7.4 supplemented with
0.01 % Tween 20 (v/v) (PBST) as running buffer
at a flow rate of 12.5 µl/min. The Biosensor
surface was primed by three injections of 100 µl
immobilization buffer. Each immobilization was
performed by activating the sensor chip surface
with a 125 µl injection of 0.2 M EDC and 0.05 M
NHS (mixed from 2x stock right before injection).
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Next, 188 µl of target protein was injected and
residual activated groups on the surface were
blocked by injecting 125 µl of 1 M ethanolamine
(pH 8.5). As a blank control, one channel was
activated and blocked without intermediate
injection of protein.
Preparation of compounds and affinity
measurements
Maltose and glucose were dissolved in PBST
(running buffer) to yield final concentration of
500 µM. A 1:1 dilution series ranging from 500 µM
to 61 nM was then applied for binding experiments
starting with the lowest concentration.
Affinity measurements were performed at 22 °C at
a flow rate of 30 µl/min using PBST as running
buffer. Analyte was injected for 5 min followed by
a 10 min wait (running buffer flow at 30 µl/min).
Data analysis
Using the FitMaster® Origin-based software raw
data were cut and analyzed:
 Subtraction of a reference channel
(channel with no protein immobilized).
 Affinity constants and Kd values were
determined from equilibrium response
values using the provided 1:1 binding
model.
Instrumentation
NanoTemper
Technologies
Seismos
NT.X
Instrument (Surface Acoustic Wave Biosensor).
NanoTemper Technologies SAW Sensor Chip
COOH-SAM.
Acknowledgement
MBP was kindly provided by Susanna v. Gronau
and Dr. Sabine Suppmann from the biochemistry
core facility of the Max Planck Institute of
Biochemistry, Martinsried.
References
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