Moss Tom. DNA-protein interactions: principles and protocols
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Calorimetry of Protein–DNA Complexes 523
9°C; trace c: eight injections of 490 µM Sox-5 into 60 µM of 12-bp DNA at 30°C; trace
d: control titration of Sox-5 into buffer at 30°C. The injection rate was 8 µL in 12 s.
The time intervals between injections were 4 min (traces a and b) and 5 min (traces c
and d). (B) The open circles represent ∆H
app
measured between 9 and 30°C and calcu-
lated according to Eq. 6 (mean values of six to eight injections). Note that ∆H
app
changes sign at about 17°C. The standard deviations from the mean values shown are
of the order of ±5–10%. Filled circles represent the binding enthalpy obtained after
correction of ∆H
app
for protein and DNA refolding and complex unfolding. The lines
correspond to linear least-squares fits to the data, the slope of which is equal to ∆C
p
.
524 Read and Jelesarov
volume of the cell, V
cell
. The enthalpy of association, ∆H
app
, at temperature T
may then be calculated from Eq. 6. This procedure is repeated for the pairs of
titrations at different temperatures. The experimentally observed enthalpies
(∆H
app
) obtained at a number of temperatures can be plotted as illustrated in
Fig. 3B.
3.2. Differential Scanning Calorimetry
1. Prior to starting experiments ensure that the Nano-DSC calorimeter sample and
reference cells are at room temperature, thoroughly cleaned, and thermally bal-
anced.
2. Add working buffer (100 mM KCl, 10 mM potassium phosphate, and 1 mM
EDTA (pH 6.0)) which is at room temperature to both sample and reference cells.
It is most important that no air bubbles are present in the calorimeter cells (see
Note 7). The presence of air bubbles will yield incorrect values for the partial
molar heat capacity of the macromolecule, because the volume of solution in the
sample and reference cells is not identical (assumed in Eq. 7). Furthermore
expansion of the minutes of air bubbles with heating results in significant heat
output (and vice versa on cooling).
3. Seal the top of the chamber with the screw-threaded piston and let the calorim-
eter settle to thermal equilibrium. Apply approx 2 atm of overpressure to the cells
by screwing down the piston. As pressure is applied note the compensatory power
reading—it should not change by more than a few microwatts (see Note 8).
4. Scan to obtain a buffer–buffer baseline. Initially, a number of scans are recorded
in order to obtain a number of reproducible baselines (to within 0.5 µW, in the
linear region). A complete cycle of heating and cooling at a rate of 1 K/min actu-
ally takes about 4 h because at the end of each heating or cooling scan, there is a
period of thermal equilibration.
5. Remove the buffer from the sample cell. This is most easily accomplished with a
vacuum line attached to a water pump.
6. Slowly and carefully apply a 1.8-mL dialyzed sample to the cell, using the method
outlined in Note 7. For concentrated protein samples, care is needed to remove
all air bubbles. The excess sample is put to good use: first to determine sample
concentration and, second, it is diluted with the final dialysate for use in further
calorimetric runs. Again, apply a pressure of approx 2 atm to the cells.
7. Perform one heating and cooling scan to obtain the denaturation and renaturation
curves of the sample-buffer. Usually samples were heated from 0°C to 80°C and
then cooled to –8°C, both at a rate of 1 K/min. The heat effects of unfolding and
refolding should appear as mirror images, slightly shifted in temperature because
the slower kinetics of refolding. Reproducibility shows that the heat-induced
unfolding/refolding is reversible. However, this may not be the case (see Note 9).
8. Remove the sample from the cell. Thoroughly wash out the cell with buffer and
then perform another scan of buffer. A heat-asorption peak may be observable if
the previous sample is not completely removed (see Note 10).
Calorimetry of Protein–DNA Complexes 525
3.2.1. DSC Data Analysis
The subtraction of buffer scans to obtain an accurate baseline, the conver-
sion to partial molar heat capacities, and the deconvolution of the excess molar
heat capacity function into separate transitions were all performed using the
CPCalc program. CPCalc provides a simple interactive mechanism, based on
Data Exchange Ports, for the parsing of data from one step to another.
1. The functions of differential power and temperature vs time are extracted from
the DSC data acquisition files. Each file may contain more than one scan, but
further analysis is on a one-scan basis.
2. A matching buffer–buffer scan is subtracted from a sample-buffer scan. Both
must be from a heating (or cooling) scan. Since the absolute values of the molar
heat capacities depend on good baseline subtraction and the buffer-buffer scans
vary to a small extent, it is important that a number of subtractions are tried using
different scans from the acquisition file.
3. The subtracted compensatory power curve is converted to the partial molar heat
capacity function by use of Eq. 7. Accurate values for the concentration (Sub-
heading 3.4.), molecular mass, and partial specific volume of the macromolecule
are required (see Note 11).
4. Appropriate functions for the intrinsic heat capacity of the initial and final states
may then be put onto the partial molar heat capacity function (Fig. 4). The
calorimetric ∆H, ∆S, and melting temperature of the peak are obtained. Enthalpies
and entropies over a restricted temperature interval, ∆H(T) and ∆S(T), are obtained
by viewing the partial molar heat capacity function between the two temperatures.
5. The excess molar heat capacity function is deconvoluted into separate transitions.
3.3. Correction of ITC Derived Enthalpies
The value for ∆C
p,app
calculated from the slope of the ∆H
app
vs T plot
(Fig. 3) is about –3.3 kJ/K/mol, implying that a considerable amount of hydro-
phobic surface is buried at the complex interface. However, over the tempera-
ture range (10–30°C) used for the ITC measurements, the partial molar heat
capacity functions for both free Sox-5 and DNA (DSC data, Fig. 4A) are greater
than the calculated intrinsic heat capacity functions for the fully folded mol-
ecules. Thus, the DSC experiments show significant heat absorption resulting
from a temperature-dependent unfolding of the free Sox-5 and DNA. Signifi-
cant refolding of both components must therefore take place when they associ-
ate at these temperatures: a simple interaction between rigid-body molecules is
not observed. If one wishes to relate observed energetic parameters to struc-
tural features of a rigid fully-folded complex, then the enthalpic contributions
arising from refolding of the free Sox-5 and DNA, as determined from DSC,
must be added to the ITC-measured enthalpies of association to obtain the
enthalpies applicable to a rigid-body interaction. Furthermore, because the
526 Read and Jelesarov
Fig. 4. (A) Partial molar heat capacity functions obtained for the free Sox-5 protein
(dotted curve), the 12-bp DNA duplex that includes the AACAAT motif (dot–dash
curve) and the 1:1 Sox-5–12-bp DNA complex (solid curve). Note that the complex
dissociates in a single cooperative trimolecular transition with a melting temperature
only slightly above that of the 12-bp DNA duplex. The free Sox-5 protein and 12-bp
DNA duplex dissociate in a monomolecular and bimolecular manner, respectively.
(B) Partial molar heat capacity function for the Sox-5–12-bp DNA complex showing
the intrinsic heat capacity functions for the fully folded (N) and unfolded (D) complex
(thin lines). The area above the dashed line corresponds to the total enthalpy of all the
temperature-induced changes in the complex.
Calorimetry of Protein–DNA Complexes 527
complex also absorbs heat over the 10–30°C temperature range (i.e., becomes
partially unfolded [Fig. 4A]), such heats must be subtracted from the ITC-
measured enthalpies of association to generate the net enthalpies applicable to
the formation of a fully folded complex. These corrections considerably affect
the enthalpy profile of binding (see Fig. 3B). The corrected plots of ∆H
app
vs T
are now linear, with an average slope of about –1.5 kJ/K/mol and this corrected
value of ∆C
p
is in very good agreement with the amount and type of molecular
surface screened from the solvent upon association, calculated assuming a
rigid-body interaction.
3.4. Determination of Concentrations
The protein and DNA samples must be highly homogenous and their con-
centrations must be known to the highest possible precision. In the first place
this enables an accurate preparation of DNA duplex from its complementary
oligonucleotides and of the Sox-5–DNA complex from protein and DNA
duplex. In DSC, the accuracy with which the sample concentration can be
determined directly affects the molar heat capacity values obtained. In ITC,
unlike other binding assays, errors in the concentration of protein are
directly reflected in the values of ∆H
app
(Eqs. 3 and 4). Thus, in both ITC and
DSC, concentrations are determined after dialysis. In the DSC experiments, it
is also possible that a small amount of buffer from a previous scan remains in
the gold capillary cell. To eliminate this potential source of error, concentra-
tions were determined using the excess sample that remains after filling the
calorimeter cell.
3.4.1. DNA
For oligonucleotides and DNA duplexes, concentrations were determined
from their UV absorption at 260 nm, after digestion to nucleotides with snake
venom phosphodiesterase I (PDE1).
1. Accurately dilute the solution of DNA to about 1.0 mL with 100 mM Tris-HCl
(pH 8.0) so as to give an absorbance at 260 nm of about 0.5 in a 1-cm pathlength
cell. To overcome any reliance on the presumed accuracy of the pipets being
used, the dilutions are performed on a precision balance.
2. Record the UV absorption spectrum of the diluted DNA solution, using as reference
a blank cell containing buffer. Note that for small oligonucleotides and DNA duplexes
with a biased nucleotide composition, the absorption maximum is not necessarily at
260 nm. This is in fact an optional step, but the recording of UV spectra both
before and after the addition of PDE1 indicates that digestion has occurred and
enables a determination of the hypochromicity of the DNA solution.
3. Remove the diluted DNA solution from the cell and place into a screw-capped
1.5-mL tube. Add either 0.008 U (for oligonucleotides) or 0.08 U (for DNA
528 Read and Jelesarov
duplexes) of PDE1 to the solution, mix well, and incubate at 37°C overnight. If
necessary, a time-course of the digestion may be followed (at room temperature)
in the UV spectrophotometer by direct addition of PDE1 to the DNA solution in
the quartz cell.
4. Accurately record the UV absorption spectrum of the DNA solution after PDE1
digestion, using a black cell containing buffer as a reference. The contribution
to the absorbance at 260 nm arising from overlap of the PDE1 280-nm peak may
be neglected
5. Optionally, calculate the percent hypochromicity (%H) from the equation
%H =
(A
a
– A
b
)
× 100
A
a
where A
b
and A
a
are the absorbances at 260 nm before and after PDE1 digestion
respectively, with dilution the result of the addition of PDE1 taken into account.
Values of %H are usually approx 20% for oligonucleotides and approx 35–40%
for duplex DNA. Lower values indicate that digestion may not be complete, that
the original DNA is degraded, or that there is a failure to form duplex DNA.
6. Calculate the molar nucleotide concentration and thus the molar DNA concentra-
tion from the following equations
[Nucleotide] =
Aa(Dilution)N
(12,010G) + (15,200A) + (8400T) + 7050C)
and
[DNA] =
[Nucleotide]
N
where N is the total number of nucleotides in the DNA and G, A, T, and C are
respectively the number of dG, dA, dT, and dC nucleotides. The extinction
coefficients at 260 nm for the four nucleotides are from ref. 23.
3.4.2. Protein
The concentration of Sox-5 (in working buffer) was determined from its UV
absorption at 280 nm using an extinction coefficient of 17,460/M/cm. This
extinction coefficient is based on the addition of the extinction coefficients of
tryptophan and tyrosine (of which there are two and five in Sox-5, respec-
tively). The accuracy of the concentration determination is within 5%.
3.4.3. Sox-5–DNA Complex
The concentration of complex was determined from its UV absorbance at
260 nm. At this wavelength, the absorption is mainly the result of the DNA,
but there is a significant absorption from the protein that must be taken into
account. There may also be additional hyperchomic effects because the DNA
of the Sox-5–DNA complex is highly bent with considerable base unstacking.
Calorimetry of Protein–DNA Complexes 529
Thus, the approach taken was to measure the UV absorbance at 260 nm for a
solution of DNA duplex and then again after the addition of an equimolar
amount of Sox-5 protein to form the complex. The measurement was performed
at approx 60 µM DNA in a 1-mm pathlength cell, such that the complex was
fully associated. For the Sox-5–12-bp DNA complex, an extinction coefficient
at 260 nm of 158.6 × 10
3
/Molar complex/cm was obtained.
4. Notes
1. The choice of assay buffer depends on the system under study and on the require-
ments of other assays performed in the context of a particular investigation. Buff-
ers with a different heat of protonation may be used in an ITC experiment to
check whether the protonation state of the system changes upon association.
2. In setting the stirring speed, the particular system under study must be consid-
ered. Some proteins do not resist the forces arising from rapid stirring of the
solution placed in the narrow calorimetric cell and aggregate or unfold. On
the other hand, very slow stirring may result in low rates of heat transfer, thus
causing broadening of the peaks observed in the thermogram and a decrease
in sensitivity.
3. When working below room temperature, it is recommended to fill the cells with
cold solutions, otherwise the equilibration time may be very long. Any particles
in the stirred sample cell as well as the formation of bubbles during filling may
cause problems in establishing the baseline.
4. There is no general rule about the number, volume, and duration of injections. To
obtain an entire binding isotherm a 2–4 molar excess of ligand over the concen-
tration of receptor binding sites should be injected by at least 10–15 additions.
Five to 10 injections suffice to reliably measure the enthalpy of reaction. In this
case, the degree of saturation must be less than 0.5 at the end of experiment. The
volume and duration of injections should be chosen in such a way that the released
heat is well above the threshold of sensitivity and sharp peaks appear in the ther-
mogram. Typically, 5-min intervals between injection are sufficiently long times
for the signal to return to the baseline. However, for very slow reactions, this
interval should be prolonged. Because the heat of reaction is a temperature-
dependent quantity, the injection scheme might be changed for experiments
carried out at different temperatures.
5. Alternating cycles of washing with 0.5 M NaOH and isopropanol are routinely
used. To remove heavily precipitated proteins, the cell can be rinsed with a hot
solution of 20% sodium dodecyl sulfate (SDS). The protocol for cleaning will
depend on the particular system under study and the material of the cell.
6. Integration requires construction of a proper baseline. Some software products
support automatic procedures for baseline determination. However, it is often
found that manual adjustment of the baseline is a better practice, particularly in
cases when the signal-to-noise ratio is low or when the instrument baseline drift
is substantial. In constructing the baseline manually, it is important to use the
530 Read and Jelesarov
same integration window for all the peaks observed, both in the specific binding
and in the blank titration experiments. This will avoid the introduction of nonran-
dom bias in the data.
7. We would advocate filling each cell using a minimum of approx 1.8 mL of solu-
tion by the following method (see also Fig. 5). First attach a 1-mL pipet tip using
a short length of silicon tubing to one port of the cell. Draw up 0.9 mL of solution
using a pipettor fitted with a 1-mL tip and silicon tubing. Connect this to the
other side of the cell and slowly introduce the solution into the cell. Disconnect
and apply a second 0.9 mL of solution, without introducing any air bubbles. As
the cell is only about 0.9 mL in volume, excess solution rises up into the 1-mL
Fig. 5. Arrangement for filling the capillary cells in the Nano-DSC calorimeter.
Calorimetry of Protein–DNA Complexes 531
pipet tip attached to the other side. The solution is then pipetted up and down, at
first slowly and then more rapidly, for sufficient time to expel minute air bubbles
from the cell. Eventually, excess solution may be removed by carefully with-
drawing it into the 1.0-mL tip connected to the pipettor and then disconnecting
by simultaneously pulling off both 1-mL tips with their connecting tubing.
8. If the microwatt reading rapidly increases or decreases on applying pressure, then
air bubbles are present in either of the cells. The cells must then be emptied
and refilled.
9. One reason may be temperature-induced aggregation, manifesting itself as a large
peak of heat evolution that typically commences close to the peak of maximum
heat absorption of the cooperative transition (approximately at the melting tem-
perature, T
m
). A s possible reason for irreversibility is the presence of disulfide
bonds in the protein, which may disrupt on heating but, on cooling form, mixed
disulfide bonds with incorrect Cys residues. This may be overcome by using
proteins (or domains) with no Cys residues (as in Sox-5) or by using engineered
versions in which the Cys residues are mutated to Ser. This strategy will, how-
ever, depend on the contribution of the disulfide bonds in stabilizing the overall
protein fold.
10. The calorimetric cells may be thoroughly cleaned by filling them with 50% (v/v)
formic acid, followed by heating from 25°C to 65°C (at 1 K/min). The cells are
then thoroughly rinsed with water and buffer. Multiple baseline scans of the
buffer are recommended before proceeding with the next sample.
11. The partial specific volumes used for oligonucleotides and DNA duplexes were
0.53 and 0.54 mL/g. The partial specific volume of Sox-5 was calculated from
its composition as 0.723 mL/g, using the known partial specific volumes of
the amino acids. The Sox-5–12-bp DNA complex partial specific volume of
0.650 mL/g was calculated as the weight average of the partial specific volumes
of Sox-5 and the DNA duplex.
Acknowledgments
We would like to thank Peter Privalov (Johns Hopkins University, Balti-
more), in whose laboratory the calorimetry was performed. We thank Colyn
Crane-Robinson for critical reading of the manuscript. Financial support from
an NIH grant to the Baltimore laboratory (GM48036-06), a Wellcome Trust
grant to the Portsmouth laboratory, and NATO Collaborative Research Grants
are gratefully acknowledged.
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