TECHNOLOGY REVIEW
Thermal Denaturation Assays in Chemical Biology
Guillermo Senisterra, Irene Chau, and Masoud Vedadi
Structural Genomics Consortium, University of Toronto,
Toronto, Ontario, Canada.
ABSTRACT
Thermal denaturation-based methods are becoming increasingly used to
characterize protein stability and interactions. Recent technical advances have made these methods more suitable for high throughput
screening. Reasonable throughput and the ability to perform these
screens using commonly used instruments, such as RT-PCR machines or
simple plate readers equipped with heating devices, facilitate these
experiments in almost any laboratory. Introducing an aggregation-based
monitoring approach as well as alternative fluorophores has allowed the
screening of a wider range of proteins, including membrane proteins,
against large chemical libraries. Thermal denaturation-based methods
are independent of protein function, which is especially useful for the
identification of orphan protein function. Here, we review applications
of thermal denaturation-based methods in characterizing protein stability and ligand binding, and also provide information on protocol
modifications that may further increase throughput.
INTRODUCTION
T
hermal denaturation-based methods have increasingly become the method of choice for screening proteins against
libraries of compounds and conditions that may stabilize
proteins. Protein thermal denaturation can easily be monitored by fluorescence- (differential scanning fluorimetry [DSF])1–4
or aggregation- (differential static light scattering [DSLS])1,5 based
methods. In DSF, the protein is heated at a controlled heating rate,
usually 1C/min, at temperatures ranging from 25C to 95C in the
presence of environmentally sensitive fluorophores. In the hydrophobic environment introduced when the protein unfolds, the fluorescence signal increases significantly.3,4,6,7 This signal can be
monitored by using a variety of plate readers, including RT-PCR devices, that are available in most university departments. Several probes
are suitable for this purpose, including 1-anilinonaphthalene-8sulfonate.7 More recently, probes with higher fluorescence quantum
yields, such as Sypro-orange, have been introduced.3,6 Alexandrov
et al. recently reported the use of the thiol-specific fluorochrome
N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl] maleimide (CPM)
for the stability profiling of membrane proteins.8 Plotting the intensities obtained from such experiments vs. temperature typically results
in sigmoid curves that can be fit by using nonlinear regression. The
point of inflection of each resulting curve is defined as the melting
temperature (Tm) (Fig. 1A). The stability of most proteins decreases
with temperature; as the temperature increases, the Gibbs free energy
of unfolding decreases and becomes zero at equilibrium where the
concentrations of folded and unfolded protein are equal. At this point,
the temperature is considered as Tm. If the protein unfolds in a reversible two-state manner, the equilibrium thermodynamics models
will apply.9 A protein can be typically screened in a 384-well plate in
the presence of up to 384 different compounds or buffer conditions.
An increase in Tm (DTm) is an indication of stabilization and reflects
compound binding (Fig. 1A). The effect of ligand binding on protein
stability is generally concentration dependent (Fig. 1B),1,10,11 and
affinity constants at the Tm can be calculated from the resulting
curves.11–13 The binding affinities measured by such an approach
often correlate with the binding affinities obtained by isothermal titration calorimetry.11 However, due to a high fluorescence background, some proteins may not be amenable to DSF screening by
using Sypro Orange.1 Using alternative probes such as CPM may be
more useful in these cases.8,14 A more detailed DSF protocol is
available.3 A video of DSF, which reviews all experimental and data
analysis steps, is also available online (www.videoprotocols.org).
In comparison, the aggregation-based thermal denaturation
method (DSLS; Harbinger’s StarGazer-384) is label free and relies
on the immediate aggregation of proteins upon unfolding.1,5 Clearbottom 384-well plates (e.g., Nunc, Rochester, NY) are used, and
incident light is shone on the protein drop from beneath at an angle
of 30. Protein samples in the presence and absence of compounds
are heated from 25C to 85C, and aggregate immediately upon
unfolding. Protein aggregation is monitored by measuring the intensity of the scattered light every 30 s by using a charge-coupled
device camera. The pixel intensities in a preselected region of each
well are integrated to generate a value that is representative of the
total amount of scattered light in that region. These total intensities
are then plotted against temperature for each sample well, similar to
DSF. The resulting point of inflection of each curve, however, is
ABBREVIATIONS: CHKA, human choline kinase isoform A; CPM, N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl] maleimide; DSC, differential scanning calorimetry;
DSF, differential scanning fluorimetry; DSLS, differential static light scattering; Epha, human ephrin receptor EphA; GDP, guanosine 50 -diphosphate; GMD, GDP-Dmannose 4,6-dehydratase; GRM, human metabotropic glutamate receptor; HDAC4, histone deacetylase 4; ITD, isothermal denaturation; PAP, 30 -phosphoadenosine 50 phosphosulfate; PKM2, pyruvate kinase M2; PLP, pyridoxal 5-phosphate; PPI, peptidyl prolyl cis-trans isomerase; Rop18, rhoptry kinase; SNP, single nucleotide
polymorphism; SULT 1A3, human cytosolic sulfotransfases 1A3; SULT 1C1, human cytosolic sulfotransfases 1C1; Tm, melting temperature; WDR5; WD40-repeat protein,
part of MLL methyltransferase complex.
128 ASSAY and Drug Development Technologies APRIL 2012
DOI: 10.1089/adt.2011.0390
THERMAL DENATURATION ASSAYS IN CHEMICAL BIOLOGY
A
B
1.0
10
0.9
0.8
6
DTm
Fluorescence
8
0.7
0.6
4
0.5
2
0.4
0
0.3
20
30
40
50
60
70
80
0
2
Temperature (°C)
4
6
8
10
GDP (mM)
Fig. 1. Detection of ligand binding by DSF. (A) Effect of GDP binding on the stability of GMD was measured by DSF at 0.1 mg/mL protein
concentration and a scan rate of 1C/min in the presence of 0 mM (black), 0.15 mM (red), 0.31 mM (green), 0.63 mM (blue), 1.25 mM
(magenta), 2.5 mM (dark yellow), 5 mM (purple), and 10 mM (orange) of GDP. (B) The DTm values at different concentrations of GDP are
also plotted to illustrate that the stabilization effect of GDP is concentration dependent. Tm values were calculated from fitting the
transition curves in (A) by using the Boltzman sigmoid function as previously described.1 GMD, GDP-D-mannose 4,6-dehydratase; GDP,
Guanosine 50 -diphosphate; DSF, differential scanning fluorimetry; Tm, melting temperature.
defined as Tagg. For most proteins, Tagg and Tm values are reasonably
close.1 However, DSLS is an indirect measurement of unfolding; and
some proteins (especially very small proteins) that do not aggregate
over short time scales immediately after denaturation may not be
amenable to DSLS. Any factors affecting the light scattering readout
will also affect the initial light scattering values and will be detected
even before the start of the experiment. The DSLS is also applicable to
proteins with high fluorescence backgrounds.15 Additionally, DSLS is
insensitive to the presence of detergents or protein hydrophobicity
and has been used to monitor the thermal denaturation of membrane
proteins, assess their stability, and detect ligand binding in a 384well format.15 ATLAS (Any Target Ligand Affinity Screen) is another thermal denaturation-based method that detects thermally
unfolded and aggregated hexahistidine-tagged proteins. This method
uses time-resolved fluorescence resonance energy transfer between
two anti-hexahistidine antibodies, which are labeled with either a
donor or acceptor fluorophore, that are simultaneously bound to the
hexahistidine tags of the aggregated protein.16,17 Advantages and
limitations using these and other thermal denaturation-based
methods have been previously discussed in greater detail.14
Differential scanning calorimetry (DSC) is also a reliable, yet lowthroughput, method for analyzing protein thermal stability.18,19
However, recently developed microelectromechanical system-based
DSC provides the possibility of using less protein and potentially
higher throughput in the future.20 The DSC measures the heat absorbed as a function of temperature at constant pressure. The shape of
the heat capacity function (Cp vs. T) provides information on the
thermodynamics of the order-disorder transition. The DSC is used to
determine the DH, DCp, and Tm of a structural change in a biological
macromolecule. Thermograms obtained by DSC can be analyzed for
protein denaturation or unfolding, from a simple two-state model
where intermediates between the initial and final states are not
significantly populated at equilibrium to a more complex, non-twostate model that involves multiple states of unfolding. In the twostate denaturation model, both native and denatured molecules are
the only assumed states. For the non-two-state model, however, two
cases are considered: one assumes the existence of several domains in
the protein, each of which unfolds independently in a two-state
manner, and the other model considers multiple, intermediate
steps.21 Other methods such as circular dichroism have also been used
to monitor protein thermal denaturation and protein stability.22–28
ATYPICAL THERMAL DENATURATION CURVES
MAY PROVIDE VALUABLE INFORMATION
Assessing the stability of proteins and detecting ligands by
fluorescence-based methods is not always easy. Fluorescence may
interfere, and changes in the shapes of transition curves can also
make it more difficult to determine and compare Tm values in the
presence and absence of compounds.14 In some cases, one may be
able to extract additional information from transition curves. As just
noted, heat capacity profiles obtained by DSC contain information
related to the unfolding partition function that can be used to evaluate the number of states populated during the transition as well as
the thermodynamic parameters associated with them.29 The denaturation of many proteins shows multiple transitions that indicate
multiple denaturation components.30–33
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SENISTERRA, CHAU, AND VEDADI
For some proteins, more than one transition can be detected in
the presence of a ligand. This may be due to the heterogeneous
solutions of bound and unbound protein molecules, especially at
low ligand concentrations. Under such conditions, one typically
expects to observe the disappearance of multiple transitions on increases in the ligand concentration when reaching saturation. For
example, in bovine a-lactalbumin when all Ca2+ -binding sites are
saturated, a single transition is observed by DSC; at concentrations
where free calcium levels are less than the protein concentration,
two heat absorption peaks are detected, and the low temperature
peak is assigned to the apo form of lactalbumin.34 It is clear from
these double transitions that Ca2+ concentrations lower than protein
Fig. 2. Atypical transition curves provide valuable information. Effect of NADP+ binding on the stability of GMD were measured by DSF at
0.1 mg/mL protein concentration and at a scan rate of 1C/min (A) at NADP+ concentrations of 0 mM (black), 0.15 mM (red), 0.31 mM
(green), 0.63 mM (blue), 1.25 mM (magenta), 2.5 mM (dark yellow), 5 mM (purple), and 10 mM (orange). (B) Effect of GDP binding in the
presence of 5 mM NADP+ on the stability of GMD was also measured by DSF at GDP concentrations of 0 mM (red), 0.63 mM (green),
1.25 mM (blue), 2.5 mM (magenta), 5 mM (dark yellow), 8 mM (purple), and 10 mM (orange). Black represents a control in the absence of
NADP+ and GDP. Differential unfolding of two domains of GMD is depicted when (C) NADP+ (shown in green) is bound to one domain but
the other domain (GDP binding domain) is unoccupied, and when (D) NADP+ is bound to one domain and GDP (shown in red) to the other.
Sypro orange, the molecular structure of which is undisclosed, is shown as a three-ring aromatic molecule. When only NADP+ binds to the
protein, NADP+ binding domain is stabilized and will denature at a higher temperature compared with unoccupied GDP binding domain as
shown in (C). However, if NADP+ binds to one domain and GDP to the other as shown in (D), both domains are stabilized and will denature
cooperatively.
130 ASSAY and Drug Development Technologies APRIL 2012
THERMAL DENATURATION ASSAYS IN CHEMICAL BIOLOGY
than the Tm values predicted for high scan rates.37 Increasing scan
rates reduces experimental time, and as long as such an increase does
not affect the sensitivity of ligand detection, it would accelerate the
compound screening process. This is fundamental when screening
large numbers of compounds, as the overall time can be reduced by
one-half by doubling the heating rate, saving days or weeks, depending on the size of the project. It is commonly accepted to use
scan rates between 0.5 and 1C/min for protein denaturation experiments using either DSC or DSF.
Measurements of Tm values for nine different proteins by using
five different heating rates (1, 2, 4, 6, and 8C/min) revealed that the
Tm values generally increase as the heating rate increases (Fig. 3).
This is consistent with the reports just mentioned. Although the increase in Tm upon increasing the scan rate is significant, it is not
dramatic and allows ligand screening by detection of the DTm. This
raises the possibility of rapid screening and is time saving. However,
the question remains as to how fast one can scan and still be able to
detect ligand binding (DTm). We, therefore, screened nine proteins
against one or two of their known ligands at various concentrations
(Fig. 4). In the majority of cases, no significant changes in DTm were
observed (Fig. 4A). Although in certain cases, the DTm values decreased, only in two cases did the DTm values fall below the 1C
70
65
60
Tm (°C)
concentrations prevent the apo and holo forms from interconverting
quickly.34
However, for some proteins, increases in the concentration of
ligand may either lead to more distinct multiple denaturation transitions or help further stabilize a ligand-binding domain that otherwise denatures at a lower Tm. This point can be explained more easily
if the ligand binds to a distinct domain in a multi-domain protein and
stabilizes the domain more than the overall protein structure. In such
cases, an increase in ligand concentration is expected to shift the
transition related to the ligand-binding domain more significantly
than the rest of the protein. For example, equine lysozyme is folded in
two domains that are separated by a cleft. Thermal denaturation of
equine lysozyme monitored by DSC shows two transitions: the first
transition is associated with the b domain, a Ca2+ -binding domain,
and the second peak is associated with the a domain. The Tm for the
Ca2+ -binding domain shifted to a higher temperature when Ca2+
levels were increased; however, the Tm for the a domain remained
unchanged. The presence of Ca2+ , therefore, reduces interdomain
cooperativity during equine lysozyme denaturation.35
Multiple transitions observed by DSF on ligand binding may also
be related to the differential stabilization of domains within multidomain proteins. For example, human GDP-D-mannose 4,6dehydratase (GMD), which has a typical DSF transition, shows a double
transition in the presence of NADP+ . Interestingly, by increasing the
concentration of NADP+ , the first transition remains unchanged, and
the second transition shifts to a higher temperature (Fig. 2A, C). The
addition of different concentrations of GDP to a mixture of GMD at a
fixed, saturating concentration of NADP+ results in a decrease in the
intensity of the first transition and a shift to a higher Tm (Fig. 2B, D).
Interestingly, GDP and NADP+ bind to different domains of GMD
(PDB ID: 1T2A), and differences in the effects of these two compounds on GMD stability may reflect that fact. If the binding of a
compound to the domain of a protein stabilizes that domain more
and the domain unfolds at a higher temperature than the rest of the
protein, then the distinct transition might show a similar difference
in stability. Although we do not want to overinterpret such screening
data, one may learn more about the binding of a compound from
changes in the unfolding transition on ligand binding. Multiple
transitions were also observed by using DSF when investigating the
stability of monoclonal antibody formulations, transitions that correlated well with DSC.36
55
50
45
40
0
2
4
6
8
10
Scan Rate (°C/min)
EFFECTS OF THE HEATING RATE ON TM
MEASUREMENTS AND THE DETECTION
OF LIGAND BINDING
The thermodynamic parameters that characterize protein unfolding and their dependence on the heating rate have been studied by
using DSC. These parameters are only meaningful for reversible
unfolding at equilibrium; however, the denaturation of most large
multi-domain proteins is either partially or completely irreversible.37
Dependence of the Tm on heating rates has been found for many
proteins.38–47 Using mathematical simulations, it has been shown
that for low scan rates, the predicted Tm values would be much lower
Fig. 3. Effect of the scan rate on Tm measurement. Tm values were
measured by DSF for SULT 1C1 (-) at 0.1 mg/mL, SULT 1A3 (C) at
0.05 mg/mL, CHKA (:) at 0.025 mg/mL, Epha (;) at 0.05 mg/mL,
GRM8 (A) at 0.05 mg/mL, HDAC4 (,) at 0.1 mg/mL, PKM2 (B) at
0.1 mg/mL, PPI (6) at 0.05 mg/mL, and Rop18 ( · ) at 0.1 mg/mL.
Experiments were performed in duplicate. SULT 1A3, human cytosolic sulfotransfases 1A3; SULT 1C1, human cytosolic sulfotransfases 1C1; CHKA, human choline kinase isoform A; Epha,
human ephrin receptor EphA; GRM, human metabotropic glutamate receptor; HDAC4, histone deacetylase 4; PKM2, pyruvate
kinase M2; PPI, peptidyl prolyl cis-trans isomerase; Rop18, rhoptry
kinase.
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A
B
12
12
10
10
DTm (°C)
DTm (°C)
8
8
6
6
4
4
2
2
0
0
0
2
4
6
8
Scan Rate (°C/min)
0
2
4
6
8
Scan Rate (°C/min)
Fig. 4. Effect of the scan rate on detecting ligand binding. Effect of increase in the scan rate on DTm on ligand binding was assessed by
testing the binding of known ligands to selected proteins. For simplicity in discussing the results, we have plotted the data in two different
sets: (A) scan rate (from 1C to 8C/min) had no significant effect on DTm on ligand binding: SULT 1C1; PAP 1 mM (-) and 0.05 mM (<),
PLP 1 mM (:) and 0.05 mM (C), SULT 1A3; PAP 0.05 mM (,) and PLP 0.05 mM (B), Epha; staurosporine 0.1 mM (=) and 0.005 mM ( ),
and another Epha inhibitor 0.1 mM (D), PPI; cyclosporin C 0.025 mM (;) and 0.005 mM ( · ), and cyclosporin A 0.005 mM (>), GRM8;
glutamic acid 1 mM (A), CHKA; kinase inhibitor KL6 0.1 mM (*) and 0.005 mM (1), and kinase inhibitor KL147 0.005 mM ( ); and (B)
proteins that showed a larger change in DTm on change in the scan rate: SULT 1A3; PAP 1 mM (D) and PLP 1 mM (C), PPI; cyclosporin A
0.025 mM (:), GRM8; an inhibitor 0.05 mM (<), and glutamic acid 0.05 mM (3), Rop18; kinase inhibitor KL24 0.1 mM ( + ) and 0.05 mM
(;), CHKA; kinase inhibitor KL147 0.1 mM (A). PAP, 30 -phosphoadenosine 50 -phosphosulfate; PLP, pyridoxal 5-phosphate.
threshold and could not be detected at higher heating rates (Fig. 4B).
Overall, increasing the heating rate to 4C/min may have little effect
on ligand detection for most proteins. However, it would be useful to
screen the protein at different scan rates before applying a high
heating rate to screen large compound libraries. To further test the
possibility of screening proteins against libraries of large number of
compounds at a heating rate of 4C/min, we screened WDR5, a
component of MLL histone methyltransferase complex, against a
library of 1,600 compounds at heating rates of 1 and 4C/min (Fig.
5A, B, respectively). Ten hits were identified by both screens. However, two compounds were identified as hits at 1C/min but not at
4C/min scan rate. Similarly, four compounds were identified as hits
at 4C/min only. These data confirm that screening at 4C/min would
have a minimal effect on detecting stabilizing compounds.
APPLICATIONS OF THERMAL
DENATURATION METHODS
Thermal denaturation methods (e.g., DSF and DSLS) have recently
been optimized for high-throughput screening applications1,2,5
(Table 1), including chemical profiling of different protein families,
identifying novel ligands,10,48–50 and investigating the stability of
large numbers of proteins in buffers with different chemical compositions.1,14,51–55 For example, DSF has been applied to the
screening of monoclonal antibody formulations. Antibodies were
132 ASSAY and Drug Development Technologies APRIL 2012
screened in many different buffers, and the elevated aggregation
levels induced by salt, pH, and high protein concentrations were
successfully assessed.36 In addition, the effects of ions on protein
stability, such as the effects of Ca2+ and Mg2+ on collapsin response
mediator protein 2 (CRMP-2), have been investigated by using DSF.52
DSF has been used in the search for buffer conditions that reduce
aggregation levels.36,42 However, initial protein aggregation level
can only be directly monitored by DSLS.1,5,22 Thermal denaturation
methods have been widely used to investigate changes in the stability
of proteins encoded by genes with single nucleotide polymorphisms
(SNPs)56 or point mutations,57 and comparing the stability of different isoforms58 or mutants of the same protein.59
Thermal denaturation-based screening has long been considered a
reliable method to identify ligands with an eye to drug discovery.1,2,4,60 In one of the first such reported applications, screening
MurF, an essential, bacterial cell wall biosynthesis enzyme, against a
library of 200,000 compounds by using ThermoFluor technology (the
same principle as DSF2) resulted in identifying 17 hits, of which one
was found to inhibit MurF activity with an IC50 value of 25 mM.61
Follow-up screening by using ThermoFluor technology in the presence and absence of ATP helped distinguish compounds that compete
with ATP. Using the same method, HDM2 (a human ubiquitin E3
ligase) was screened against *400,000 compounds.62 With a hit rate
of <0.4%, potent HDM2 binders were identified. HDM2 binds to p53,
THERMAL DENATURATION ASSAYS IN CHEMICAL BIOLOGY
inhibiting its tumor suppressive function. The DSF has also been
employed to screen 156 kinase inhibitors against 60 human Ser/Thr
kinases to determine specificity and cross-reactivities of these in69
68
hibitors.49 Such reports confirm that thermal denaturation-based
67
screening methods can be used to screen proteins against a high
66
number of compounds in both a time- and cost-effective manner
65
64
with low false-positive rates. Recently, both DSF and DSLS were used
63
in parallel to an activity-based screening approach to rank histone
62
methyltransferase G9a inhibitors to both identify a chemical probe
61
60
for this protein and assess inhibitor selectivity within families of
0
600
1200
1800
proteins.63,64 These efforts provided valuable information on selecCompound
tivity of UNC0638 as a chemical probe for G9a.65
Ligand screening that used thermal denaturation-based methods
B (4°C / min)
75
had helped to explain the biology of proteins. For example, the
74
use of DSC showed that the binding of Mg2+ and Zn2+ increased
73
the thermal stability of p53. It was later shown that metal ion bind72
71
ing caused a conformational change and increased the surface
70
hydrophobicity of the protein.66 Screening proteins with unknown
69
68
functions against physiologically relevant compounds is a way to
67
decrypt their possible biological function. Thermal denatur66
65
ation methods are unique in such cases, as the assay is performed
0
600
1200
1800
without knowing protein function. Using ThermoFluor technology,
Compound
Carver and co-workers assigned function (nucleoside diphosphoketo-sugar aminotransferase) to an unknown but essential protein
Fig. 5. Screening at 4C/min. WDR5 was screened against a library
from Streptococcus pneumoniae after identifying pyridoxal phosof 1,600 compounds at (A) 1 and (B) 4C/min. The hits that were
phate
and pyridoxamine phosphate as high-affinity ligands of
detected by both screens (B) and those that were identified only
the
protein
by screening the protein against a library of 3,000
by one screen and not the other (,) are specified in each screen.
compounds.10
Compounds were screened at 100 mM, and they were considered
hits when they stabilized the protein by >2C. WDR5, WD40-repeat
Decreases in protein stability and susceptibility to aggregation
protein, part of MLL methyltransferase complex.
have been thoroughly investigated and linked to many diseases.
Numerous mutation-linked diseases have
a common feature: the deposition of
Table 1. Applications of High Throughput Thermal Denaturation Methods
misfolded protein aggregates. Amyloid
formation is associated with many neuApplication
Method
Reference
rodegenerative diseases, such as Alzhei1–4,48,49,61–63
Detecting ligand binding
DSF/DSLS
mer, Parkinson, and Huntington disease.67
48–51
Chemical profiling of protein families
DSF/DSLS
The thermal stability of proteins implicated in Alzheimer disease has been as1,14,36,42,51–55,74
Screening for optimum buffer conditions that stabilizes the protein,
DSF/DSLS
sessed by monitoring protein thermal
possibly reduces aggregation, or promotes crystallization
denaturation using Sypro-orange. CRMP36,77
2 has been characterized as a constituent
Antibody formulation
DSF/DSLS
of neurofibrillary tangles in Alzheimer
56,58,59
Assessing the effect of mutations and characterizing
DSLS/DSF
disease. Results from thermal stability
protein variants
assays helped identify that both Ca2+ and
10
Mg2+ affect CRMP-2 stability and prevent
Assigning function to unknown proteins
DSF
the formation of b-aggregates upon
52,56,57,73
Assessing protein destabilization associated to disease
DSF/DSLS/ITD
heating.52 Assessing the stability of 46
8,15
proteins encoded by nonsynonymous
Investigating the stability of membrane proteins and detecting
DSLS/DSF
SNPs of 16 different human proteins by
ligand binding
DSLS revealed significant differences in
76
Monitoring protein-protein interaction
DSF/DSLS
the stability of almost one-half of the
DSF, differential scanning fluorimetry; DSLS, differential static light scattering; ITD, isothermal denaturation.
variants (48%) compared with their wildtype counterparts. The half-lives of all
Tm (°C)
Tm (°C)
/ min)
A (1°C
70
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SENISTERRA, CHAU, AND VEDADI
mutants with lower stability were altered as was determined by isothermal denaturation (ITD).68 Activities of proteins encoded by 14 of
these SNPs were also significantly altered. The report also indicated
that the differences in the stability of SNPs of pyruvate kinase muscle
2 (critical for rapid cancer cell growth69,70) correlated with the decrease in their catalytic efficiencies.56
In addition, a decrease in protein stability plays a critical role in
many other diseases, such as distal arthrogryposis, a severe congenital disorder of skeletal muscle tissue.71 Tropomyosin is a key
participant in the Ca2+ regulation of muscle contraction.72 The effect
of an Arg91Gly mutation, known to be associated with distal arthrogryposis, on the stability of this protein in both skeletal and
smooth muscle has been investigated by using DSC. Previous functional analyses indicated that this mutation results in heightened
actomyosin ATPase activity at all Ca2+ concentrations. A thermal
denaturation study showed that the Arg91Gly mutation lowers the
stability of the N-terminal domain of both isoforms of tropomyosin
but does not affect the C-terminal domain.57 Changes in protein
stability by mutations or within isoforms can be assessed by using
thermal denaturation-based methods. However, the detection of
small differences may sometimes be more difficult with certain
proteins. An ITD method to assess protein stability has also been
optimized in a 384-well format and has been reported to be very
sensitive for the detection of small stability differences.58,68,73 In such
cases, protein unfolding is monitored over time at a fixed temperature (i.e., a few degrees below the Tm).
Within structural genomics efforts, compounds identified from
high-throughput screening of recombinant proteins by DSF or DSLS
often improved crystallization or crystal quality and resulted in an
increase in the rate of protein structure determination by >10%.1
Using buffers and additives identified by DSF, Ericsson et al. reported
a twofold increase in the number of crystallization leads compared
with screening in the absence of the additives.54 Crystals of fulllength CrgA from pathogenic Neisseria meningitidis MC58 were
obtained after buffer screening by using a thermal shift assay.74
Such buffer optimization has also improved purification yield and
protein quality.75
Kopec and Schneider76 recently reported the application of DSF
and DSLS to study thermal denaturation of protein-protein complexes. Interestingly, the majority of protein complexes studied
showed typical profiles usually observed for single proteins consistent with a two-state model of thermal denaturation. Similar observation was previously reported for multi-domain proteins that
showed single melting transition consistent with cooperative unfolding of domains.2 This suggests that complex formation provides
additional stabilization, and these complexes start to disassemble at
temperatures higher than the melting points of its individual components. The single melting points observed for these protein complexes were the temperature of dissociation of the complexes.
However, for some complexes, two distinct transitions were observed
that can be interpreted as differential denaturation of components of
the complexes. Alternatively, these two transitions can be related to
disassembly of the complex and melting of the components there-
134 ASSAY and Drug Development Technologies APRIL 2012
after. In any case, DSF and DSLS can be used to monitor proteinprotein complex formation in different buffer conditions.
CONCLUDING REMARKS
In this article, we tried to provide an overview of the applications
of thermal denaturation-based methods and highlight the option of
changing the heating rate that can significantly increase the
screening throughput. We also provided a case study that shows that
atypical thermal denaturation may provide additional information
which may often be ignored as screening artifacts. Overall, fluorescence- and aggregation-based thermal denaturation methods are
convenient and cost-effective ways to screen proteins against large
libraries of compounds. Assessments of protein stability have also
been successfully used to assess protein-protein interaction and to
find optimum buffer conditions that reduce aggregation and precipitation. Screening enzymes against libraries of compounds in the
presence and absence of cofactors also provides additional information on the mechanism of ligand binding. Thermal denaturationbased screening methods are particularly valuable for screening
proteins with cryptic enzymatic activity as well as those involved in
protein-protein interactions, as these methods can be used without
any knowledge of protein function. Similarly, these screening
methods are the most valuable for the identification of orphan protein function. Such screening has been optimized in a 384-well
format with a heating rate of 1C/min. However, the heating rate can
be increased to 4C/min without compromising the sensitivity of the
method for the detection of ligand binding, thereby increasing
throughput by fourfold.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Cheryl Arrowsmith for a
critical review of the article and Dr. Patrick Finerty for technical
assistance. They also thank Drs. Sirano Dhe-Paganon, Bum-Soo
Hong, Wei Qiu, Juan Carlos Pizarro, and Michael Sheun from SGC
Toronto for providing purified protein. The Structural Genomics
Consortium is a registered charity (number 1097737) that receives
funds from the Canadian Institutes for Health Research, the Canadian
Foundation for Innovation, Genome Canada, through the Ontario
Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut
and Alice Wallenberg Foundation, the Ontario Innovation Trust, the
Ontario Ministry for Research and Innovation, Merck & Co., Inc., the
Novartis Research Foundation, the Swedish Agency for Innovation
Systems, the Swedish Foundation for Strategic Research, and the
Wellcome Trust.
DISCLOSURE STATEMENT
No competing financial interests exist.
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Address correspondence to:
Masoud Vedadi, PhD
Structural Genomics Consortium
University of Toronto
101 College St.
Room 839
Toronto M5G 1L7
Ontario
Canada
E-mail: mvedadi@uhnres.utoronto.ca