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Advances in the Analysis of Conformational Transitions
in Peptides Using Differential Scanning Calorimetry
Werner W. Streicher and George I. Makhatadze
Differential scanning calorimetry can measure the heat capacity of a protein/peptide solution
over a range of temperatures at constant pressure, which is used to determine the enthalpy function of the system. There are several experimental factors that can have a significant impact on
the determined enthalpy and subsequent derived thermodynamic parameters. These factors are
discussed in terms of sample and instrument preparation, as well as data collection and analysis.
Key Words: Conformational transitions; thermodynamics; heat capacity; differential scanning calorimetry; peptides; proteins.
1. Introduction
Heat affects accompany polypeptide folding/unfolding, as any other chemical
reaction. These heat affects can be measured directly using differential scanning
calorimetry (DSC). More specifically, the enthalpy function of the system can
be determined by measuring the heat capacity at constant pressure, Cp, over a
range of temperatures.
DSC operates in differential mode, which means that the heat capacity of the
protein in aqueous solution is measured relative to the heat capacity of buffer.
Ideally, when the heat capacities and volumes of both sample and reference
cells are identical, a single peptide–buffer scan will be sufficient. However, in
reality, the sample and reference cells are slightly different and this difference
has to be taken into consideration by recording a buffer–buffer scan. Thus, prior
to starting the peptide–buffer scan, it is very important to establish baseline
reproducibility, i.e., its relative position and shape.
Figure 1 shows a typical DSC profile for a two-state unfolding process. The
area under the heat capacity profile represents the enthalpy of unfolding, the
From: Methods in Molecular Biology, vol. 350: Protein Folding Protocols
Edited by: Y. Bai and R. Nussinov © Humana Press Inc., Totowa, NJ
Streicher and Makhatadze
Fig. 1. Typical calorimetric profile (O) fitted according to a two-state model (solid-line).
Also shown are the partial molar heat capacities of the native, Cp,N, and unfolded states,
Cp,U, (dashed lines), the progress heat capacity, FN • Cp N + FU • Cp U (dotted line), the
excess heat capacity, C exc
p experimental (䊐) and fitted (solid line). The thermodynamic
parameters obtained as a result of the fit to a two-state model are ΔH = 200 kJ/mol, Tm =
65°C, ΔCp = 3.5 kJ/(mol • K).
temperature at the maximum of the excess heat capacity profile is the transition
temperature, and the difference in the heat capacities of the native and unfolded
states defines the temperature dependence of the enthalpy and entropy functions,
and, thus, the temperature dependence of peptide stability.
DSC also provides information about the modes of peptide unfolding, i.e.,
whether the unfolding process is two-state or multi-state. The effective enthalpy
of transition, usually referred to as the van’t Hoff enthalpy, is indicated by the
sharpness of the heat capacity profile. The ratio of the experimental calorimetric
enthalpy and the van’t Hoff enthalpy provides information about the mode of
the observed transition. A ratio equal to one indicates that the observed transition
is a two-state process, proceeding from the native to the unfolded state without
a significant population of intermediates (see Fig. 1). Deviation from unity indicates
that the transition is more complicated (1,2).
Recently, DSC was employed to analyze the thermodynamic properties of
short monomeric peptides that form isolated α-helices (see Fig. 2A; [3,4])
or β-hairpins, in aqueous environments (see Fig. 2B; [5]). In the case of the
α-helical peptide, the unfolded state is highly populated at temperatures higher
Analysis of Conformational Transitions in Peptides Using DSC
than 90°C, whereas the helical state never becomes fully populated, even at low
temperatures. In the case of the β-hairpin, the opposite is observed. The unfolded
state is not fully populated even at 115°C, owing to its high transition temperature, whereas the folded state is well defined at low temperatures. The major challenge for these scenarios is to define the native and unfolded state baselines for
the α-helical and β-hairpin peptides, respectively. The issue of the native-state
baseline of the α-helical peptide can be solved by assuming that the heat capacity
difference between the native (fully helical) and unfolded (coiled) states is very
small (3,4). In this case, the heat capacities of the native and unfolded states are
the same, which allows the temperature dependence of the unfolded state heat
capacity to be calculated from the amino acid composition (6). The area between
the experimental and calculated heat capacities represents the heat of helix
unfolding. This heat can be normalized for the amount of helical structure at 0°C
(estimated from circular dichroism or nuclear magnetic resonance experiments),
to give the enthalpy of helix–coil transition. For the β-hairpin peptide, the unfolding
transition appears to follow a two-state model, which allows the unfolded baseline
to be obtained by fitting the data (5). The heat capacity for the unfolded state
compares well with the heat capacity calculated using the amino acid composition of the peptide (4.6 ± 0.25 kJ/(mol • K) and 4.4 ± 0.2 kJ/(mol • K) at 75°C,
respectively), further supporting the validity of the approach (see Fig. 2).
2. Materials
1. The DSC instruments designed to study biological systems are extremely sensitive
and require small amounts of the material, 0.1–1.0 mg/mL of peptide/protein solution. Currently there are two commercial DSC instruments available, Nano-DSC
from Calorimetric Science Corporation (Provo, UT) and VP-DSC from Microcal
Inc. (Northhampton, MA). These instruments are fully automated for control, data
collection, handling, and data analysis using personal computers. The VP-DSC is
supplied with the ORIGIN graphics software.
2. A syringe with a precut needle is used to wash and load the cells (provided by the
manufacturer). The needles are precut to a specific length so that the tip of the needle
is barely above the bottom of the cell, when the overflow reservoir is in place.
3. Spectrophotometer and quartz cuvets for the cases in which the protein/peptide
concentration is determined using ultraviolet spectroscopy. It is important to consider peptide quantitation at the peptide design stage and to include aromatic
residues, if feasible, for this purpose (see Note 1).
4. Dialysis bags with the appropriate molecular weight cut-off (depending on the
protein/peptide molecular weight).
5. Highly pure protein/peptide sample. Chemically synthesized peptides must be
purified by reverse-phase high-performance liquid chromatography, which is typically performed in the presence of trifluoroacetic acid (TFA). To remove the
TFA from the purified peptide, it is recommended that the lyophilized peptide be
resuspended in deionized water and lyophilized, and the process repeated.
Streicher and Makhatadze
Fig. 2. Temperature dependence of the partial molar heat capacity. (A) α-Helical peptide AEARA6 (4). (B) β-Hairpin peptide trpzip4 (5). The thick dashed lines on both panels represent the heat capacity of the fully unfolded state calculated as described in ref. 6:
Ccalc (T ) = n ⋅ Cˆ (T ) + ( N – 1) ⋅ C – CHCONH – (T ) where N is the total number of
p ,U
p ,i
amino acid residues in the sequence, ni is the number of i-th type amino acid
residue, Ĉ p,i(T) is the partial molar heat capacity for the side chain of the i-th type,
and Cp′–CHCONH–(T) is the partial molar heat capacity for the peptide unit. The values of Ĉ p,i(T) and Cp′–CHCONH–(T) have been derived for all twenty amino acid
residues and for the peptide unit from the experimentally measured partial molar
heat capacities of model compounds for a broad temperature range (6). (B) Also
shows a fit to a two-state model using Eqs. 2–9 with ΔH = 95 kJ/mol, Tm = 78°C,
ΔCp = 0.8 kJ/(mol • K).
Analysis of Conformational Transitions in Peptides Using DSC
6. Buffer selection is of particular importance. Certain buffering components, with
high enthalpies of ionization, could change the pH significantly as a function of temperature (7). Buffers such as glycine (pH 2.0–3.5), sodium acetate (pH 3.5–5.0),
sodium cacodylate (pH 5.5–7.0), and sodium phosphate (pH 6.0–7.5), are recommended (see Note 2).
3. Method
3.1. Instrument Preparation
1. The instrument should be turned on at least 12 h prior to the experiment and “thermal history” established for the particular experimental conditions, by running
several baseline scans with the cells filled with appropriate buffer.
2. One of the most important user-defined parameters is the heating rate. Several considerations must to be taken into account. First, the increase in sensitivity is linear
with respect to the heating rate, i.e., the sensitivity with a heating rate of 120°C per
hour is twice that at 60°C per hour. One needs to keep in mind that the increase in
sensitivity actually leads to the decrease in the signal-to-noise ratio. Second, if the
expected transition is very sharp, e.g., occurs within a few degrees, a high heating
rate will distort the shape of the heat absorption profile and lead to an error in the
determination of all thermodynamic parameters for this transition and, in particular, the transition temperature. Third, the higher the heating rate, the less time the
system has to achieve equilibrium. For slow unfolding/refolding processes it is preferred to use low heating rates. Usually small globular proteins and monomeric
peptides exhibit fast folding/unfolding, and heating rates of 90–120°C per hour are
acceptable. For larger proteins it is customary to use lower heating rates (30–60°C
per hour). For fibrillar proteins, such as collagen or myosin, which exhibit very narrow transitions, a heating rate of 10–20°C per hour is more suitable.
3. The cells should be cleaned routinely (see Note 3).
4. Calibration of the instrument should be done periodically (once a year) using the
procedure provided by the manufacturer.
3.2. Sample Preparation
1. Purified protein/peptide should be extensively dialyzed with several changes of
buffer, every 6 h or more, against the appropriate buffer.
2. Prior to the experiment, insoluble particles should be removed by centrifugation at
13,000g. Filtration of the protein solution is not recommended.
3. Measure protein/peptide concentration (see Note 1).
3.3. Data Collection
1. Thoroughly wash both cells with buffer from the last dialysis change, and fill them
with the same buffer without introducing air bubbles into the cells. To achieve this,
all air bubbles should be removed from the syringe before loading. After inserting
the needle into the calorimetric cell, allow the plunger to lower slowly until the
solution appears in the overflow reservoir. At this point, start vigorously pumping
Streicher and Makhatadze
a small volume of the solution in and out of the cell. This vigorous pumping
should dislodge trapped air bubbles from the cell.
2. Fill both cells, as in step 1, with buffer and run a buffer–buffer scan.
3. After a stable baseline has been achieved, refill the sample cell with protein/peptide
solution and run a sample–buffer scan.
4. Rescan to check the reversibility of unfolding (see Note 4).
3.4. Data Analysis
1. Subtract the buffer–buffer scan from the peptide/protein–buffer scan to obtain
p (T), the heat capacity difference between sample and reference cells at
temperature T.
2. Convert ΔCapp
p (T) into the partial heat capacity of the peptide/protein at tempexp (T) as:
erature T, ΔC p,pr
C pexp
, pr (T ) =
C p , H 2O
⋅ Vpr
ΔC papp (T )
m pr
where V pr is the partial volume of the peptide/protein, mpr is the mass of the
petide/ protein in the calorimetric cell, V H O (T) is partial molar volume of aque2
ous buffer, and Cp,H O is the heat capacity of aqueous buffer. The partial volume
of the peptide/protein, V pr, can be calculated from the amino acid composition of
the protein using an additivity scheme as describe (8). The parameter Cp,H O ⲐV H O
can be considered independent of temperature and equal to 4.2 J/(K × cm–3).
3. Depending on the peptide/protein, the partial specific heat capacity of the native
state, Cp,N, at 25°C ranges from 1.25 to 1.80 J/K × g (9). The dependence of Cp,N
on temperature appears to be a linear function, with a slope from 0.005 to 0.008
J/K–2 g, which is also protein dependant (9). The partial specific heat capacity
of the unfolded state, Cp,U, is always higher than the heat capacity of the native
state. At 25°C, Cp,U values for different proteins range from 1.85 to 2.2 J/K × g,
whereas at 100°C, Cp,U values are higher, from 2.1 to 2.4 J/K × g (9). Partial heat
capacity of the unfolded state has a nonlinear dependence on temperature (e.g.,
ref. 10). It increases gradually (with the slope comparable to that for the native
state) and approaches a constant value at 60–75°C. The heat capacity change
upon peptide/protein unfolding, ΔCp = Cp,U – Cp,N, appears to be a temperaturedependent function. However, this dependence is weak in the temperature range
of 0 to 70°C, so in a first approximation, ΔCp can be considered constant.
4. Analysis of the DSC profiles, according to a certain model, can be done using the
ORIGIN software from Microcal Inc. Alternatively, any nonlinear regression software (e.g., NONLIN, NLREG, SigmaPlot, KaleidaGraph) can be used to write userdefined scripts (11). An overview of the analysis of the complex non-two-state
transitions is available (1). The following formalism is to be used for the simplest case
when the unfolding is a monomolecular two-state process (see Fig. 1; see refs. 2,12).
The heat capacity functions for the native and unfolded states are represented by
the linear functions of temperature, T, expressed in Kelvin as:
Analysis of Conformational Transitions in Peptides Using DSC
CP.N (T) = AN • (T – 273.15) + BN
CP.U (T) = AU • (T – 273.15) + BU
The equilibrium constant of the unfolding reaction, K, is related to the Gibbs
energy change upon unfolding as:
⎛ ΔG ⎞
K = exp ⎜ –
⎝ RT ⎟⎠
The Gibbs energy of unfolding, ΔG, is defined as
ΔG =
Tt – T
⎛T ⎞
ΔH fit (Tt ) + ΔC p ⋅ (T – Tt ) + T ⋅ ΔC p ⋅ In ⎜ t ⎟
where ΔCp is the heat capacity change upon unfolding taken to be independent of
temperature, Tt is the transition temperature, and ΔHfit(Tt) is the enthalpy of
unfolding at Tt. The transition temperature is defined as the temperature at which
the populations of the native, FN, and unfolded, FU, proteins are equal. The populations are defined by the equilibrium constant as:
FN (T ) =
and FU =
1+ K
1+ K
The experimental partial molar heat capacity function, C
– p,pr(Tt), is fitted to the following expression:
C p. pr (T ) = FN (T ) ⋅ CP . N (T ) + C pexc (T ) + FU (T ) ⋅ CP .U (T )
The excess heat capacity defined Cpexc(T) as:
C pexc (T ) =
ΔH (T )2
R ⋅T 2
⋅ (1 +KK )
where the enthalpy function is defined as ΔH
– (T)
ΔH(T) = ΔHfit(Tt) + ΔCp . (T – Tt)
There are seven fitted parameters: Tt, ΔH
– fit, ΔCp, AN, AU, BN, BU.
In order to analyze the data according to the above equations, the reversibility of
unfolding reaction should be established experimentally by reheating the sample.
If more than 80% of the original signal is recovered, then the reaction can be considered to be reversible. For the analysis of the irreversible transitions, see ref. 13.
4. Notes
1. Protein/peptide concentration is a very important parameter as it is required for
quantitative analysis according to Eqs. 2–9. The extinction coefficient can
be calculated from the number of aromatic residues and disulfide bonds in a peptide/protein using the following empirical equation (14):
Streicher and Makhatadze
ε 0280
= (5690 ⋅ NTrp + 1280 ⋅ NTyr + 120 ⋅ N SS ) //Mw
where Mw is the molecular mass of the peptide/protein in Daltons. A simple
experimental procedure for estimating the extinction coefficient is described (15).
Alternatively, a method based on the absorption of light at 205 nm by the peptide
bond can be used, as it is independent of amino acid composition (16).
2. The change in pH, as a function of temperature, could lead to linked protonation
effects between the buffer components and the protein/peptide. The main criterion
for using the recommended buffers is that their ionization enthalpies are similar to
those of the ionizable groups in the protein/peptide. Consequently, the deprotonation/
protonation reactions should have little or no contribution to the overall enthalpy
of unfolding. However, the change in ionization of the protein/peptide could lead
to differences in the thermal unfolding process. This effect can be investigated by
using buffer components that buffer in the same pH range, but, that have different
enthalpies of ionization (17).
3. The DSC cell should be cleaned regularly. This can be accomplished in most cases by
filling the cells with 10% SDS and heating it up to 100°C followed by a thorough rinse
with distilled water. Alternatively, the cells can be washed with 200 proof ethanol
followed by washing with distilled water. Drying the cells is not recommended.
4. The reversibility of unfolding strongly depends on the upper temperature limit during
the first scan. At high temperatures, irreversible modifications of proteins/peptides
can occur (for example, hydrolysis, deamidation, and so on; see ref. 18).
This work was supported by a grant RO1-GM54537 from the National
Institutes of Health.
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Analysis of Conformational Transitions in Peptides Using DSC
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