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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00437.x |
Applications of isothermal
titration calorimetry in protein science
Yi Liang*
State Key
Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan
430072, China
Received: May 3,
2008
Accepted: May 26,
2008
This work was
supported by grants from the National Key Basic Research Foundation of China
(No. 2006CB910301), the National Natural Science Foundation of China (No.
30770421) and the Program for New Century Excellent Talents in University (No.
NCET-04-0670)
*Corresponding
author: Tel/Fax, 86-27-68754902; E-mail, [email protected]
During the
past decade, isothermal titration calorimetry (ITC) has developed from a
specialist method for understanding molecular interactions and other
biological processes within cells to a more robust, widely used method.
Nowadays, ITC is used to investigate all types of protein interactions,
including protein-protein interactions, protein-DNA/RNA interactions, protein-small
molecule interactions and enzyme kinetics; it provides a direct route to the
complete thermodynamic characterization of protein interactions. This review
concentrates on the new applications of ITC in protein folding and misfolding,
its traditional application in protein interactions, and an overview of what
can be achieved in the field of protein science using this method and what
developments are likely to occur in the near future. Also, this review
discusses some new developments of ITC method in protein science, such as the
reverse titration of ITC and the displacement method of ITC.
Keywords isothermal titration calorimetry; protein folding; protein
misfolding; protein interaction; thermodynamics
Isothermal titration calorimetry (ITC), which provides a direct
route to the complete thermodynamic characterization of protein interactions,
has been one of the fastest developing techniques in protein science research
in the past decade [1–4]. A syringe of ITC containing a ligand is titrated into a cell
containing a protein solution. As the two elements interact, heat is released
or absorbed in direct proportion to the amount of binding that occurs. When the
protein in the cell becomes saturated with the added ligand, the heat signal
diminishes until only the background heat of dilution is observed. Measurement
of this heat allows for the accurate determination of binding constants (Kb), reaction stoichiometry (n), and a thermodynamic profile of
the protein interaction that includes the observed molar calorimetric enthalpy
(DHobs), entropy (DSobs), heat capacity (DCp,obs) of binding and change in free energy (DG). Unlike other methods, ITC does not require immobilization and/or
modification of proteins since the absorption or production of heat is an
intrinsic property of virtually all biochemical reactions [1–4].
There are at least four reasons for the increasing popularity of ITC
in the field of protein science: (1) the technique is relatively easy to
perform, resulting in the generation of a large amount of thermodynamic data
with only a small amount of protein; (2) in some instances, the Kb values of a series of protein interactions are similar or
indistinguishable [5], however, the determination of the DH and DS terms allows a further level of discrimination;
(3) although it is not possible to provide a full thermodynamic-structure
correlation of proteins from an ITC experiment, it is possible to reach
sensible conclusions from the data by comparing subtle conformational changes
of proteins; (4) the correlation of the DCp term with the change in surface area buried on forming a protein
interface has proven to be a useful tool in understanding protein interactions
with respect to both structure and thermodynamics [6,7]. ITC experiments
performed at different temperatures provide an accurate, direct determination
of the DCp term. Thanks to the recent development of
commercially available high-sensitivity instruments, for example the VP-ITC and
iTC-200 titration calorimeters from MicroCal, there has been a revival of ITC
in the field of protein science, which will help to provide a better
understanding of the mechanisms for protein interactions in signal transduction
[1–4].
Nowadays, ITC is used to investigate all types of protein
interactions, including protein-protein interactions, protein-DNA/RNA
interactions, protein-small molecule interactions and enzyme kinetics, and it
provides a direct route to the complete thermodynamic characterization of
protein interactions. The following reviews the new applications for ITC in
protein folding and misfolding, as well as its traditional application in
protein interactions. Additionally, this review provides an overview of what
can be achieved in the field of protein science using this method and what
developments are likely to occur in the near future. Some new ITC developments
in protein science, such as the reverse titration of ITC and the displacement
method of ITC, are also discussed.
ITC Applications in Protein
Folding and Misfolding
Although the principles that govern the folding of protein chains
have been widely discussed since the pioneering studies of Anfinsen [8],
knowledge about the thermodynamics of protein folding and misfolding from ITC
is relatively limited. By virtue of its general applicability and high
precision, ITC is a powerful tool for studying both the thermodynamic and
kinetic properties of protein folding. This method combined with other
biophysical methods has yielded some useful thermodynamic data on protein folding,
assembly and misfolding [9–19]. Currently, ITC is being used to solve problems related to the
important factors and the mechanisms involved in the formation and stability of
amyloid fibrils in medical research. It is also being used to directly describe
the thermodynamic properties of the folded form and the amyloid form of
proteins [2,9,14].
In a previous study, my laboratory used ITC to examine the unfolding
of rabbit muscle creatine kinase (MM-CK) induced by acid [9]. The results
indicated that the unfolding of MM-CK under such conditions is driven by a
favorable enthalpy change but with an unfavorable entropy decrease at lower
temperatures (15 ºC) and becoming entropy-driven at higher temperatures (25 ºC,
30 ºC, and 37 ºC). The increase in DconfHm, with increasing temperatures at pH 3.5 or 4.0, indicates that
thermal unfolding occurs. The changes in enthalpy and entropy for the unfolding
strongly depend on the temperature, whereas the Gibbs free energy change
happens almost independent of temperature. The enthalpy change for the
unfolding is almost compensated for by a corresponding change in entropy that
results in a smaller net Gibbs free energy increase. That is, remarkable
enthalpy-entropy compensation occurs in the acid-induced unfolding of the protein,
suggesting that water reorganization is involved in the unfolding reaction. The
value of DUGm0 for the unfolding of MM-CK induced
by guanidine hydrochloride (6.24 kcal∙mol–1) is 2-fold of that induced by acid (3.37 kcal∙mol–1) [9,10], indicating that the protein is unfolded to a greater
extent when induced by guanidine hydrochloride than when induced by acid [9].
Combining the results from ITC and other biophysical methods, we concluded that
the acid-induced unfolding of MM-CK follows a three-state model and that the
intermediate state of the protein is a partially folded monomer [9].
Isothermal acid-titration calorimetry (IATC) is a new method for
evaluating the pH dependence of protein enthalpy. In a recent publication by
Nakamura and Kidokoro [11], the enthalpy change accompanying the reversible
acid-induced transition from the native to the molten-globule state of bovine
cytochrome c was directly evaluated by this method. The results of the
global analysis of the temperature dependence of the excess enthalpy from 20 ºC
to 35 ºC have demonstrated that the native to molten-globule transition is a
two-state transition with a small heat capacity change. Since protons naturally
ligate to protein molecules [9], this new method is expected to be applicable
to the thermodynamic evaluation of the stability of many kinds of proteins,
without requiring temperature increases and without buffering reagent or
denaturant [11].
The co-chaperonin protein 10 (cpn10) is a ring-shaped heptameric
protein that exists in all organisms and whose function in vivo is to
assist cpn60 in the folding of some non-native proteins. Luke and
Wittung-Stafshede studied the assembly and disassembly of the Escherichia
coli cpn10 (GroES) and Aquifex aeolicus cpn10 in the folded state
using ITC and other biophysical methods [12]. Thermodynamic analysis revealed
that A. aeolicus cpn10’s stability profile is shifted upwards, broadened
and moved horizontally to higher temperatures, as compared to that of GroES,
and that cpn10’s higher stability originates almost exclusively from increased
monomer stability. Their study showed that protein biophysics can vary
significantly among proteins with structural homology, and at the same time, it
demonstrated that protein thermostability can be acquired without major changes
in molecular properties [12].
Protein misfolding is of intense medical interest because it is
associated with serious diseases, such as Alzheimer’s disease, Parkinson’s
disease, transmissible spongiform encephalopathy and Huntington’s disease [13].
Kardos et al have reported for the first time a direct thermodynamic
study of amyloid formation using ITC [14]. In the study, b2-microglobulin,
a protein responsible for dialysis-related amyloidosis, was used for extending
amyloid fibrils in a seed-controlled reaction in the cell of the calorimeter.
The enthalpy and heat capacity changes of the reaction, where the monomeric,
acid-denatured molecules adopt an ordered, cross-b-sheet structure in the
rigid amyloid fibrils, were investigated. Despite the dramatic difference in
morphology, b2-microglobulin has exhibited a similar heat capacity change upon
amyloid formation as that of the folding on the native globular state, whereas
the enthalpy change on the reaction has proved to be markedly lower. In
comparison with the native state, the results outline the important structural
features of the amyloid fibrils: a similar extent of surface burial even with
the supramolecular architecture of amyloid fibrils, a lower level of internal
packing, and the possible presence of unfavorable side chain contributions
[14]. More importantly, in the absence of structural information on amyloid
fibrils, the strategy used by Kardos et al in studying the thermodynamic
formation of amyloid fibrils of b2-microglobulin may become widely used in examining other protein
systems in the formation of amyloids [14].
We studied the oxidative refolding of reduced, denatured hen
egg-white lysozyme (HEL) in the presence of a mixed macromolecular crowding
agent containing both bovine serum albumin (BSA) and polysaccharide from a
physiological point of view [15]. Both the refolding yield and the rate of the
oxidative refolding of lysozyme in these mixed crowded solutions with suitable
weight ratios were higher than those in single crowded solutions, indicating
that mixed macromolecular crowding agents are more favorable to lysozyme
folding and can be used to reflect the physiological environment more
accurately than single crowding agents [15,16]. We further investigated the
effects of two single macromolecular crowding agents, Ficoll 70 and BSA, and
one mixed macromolecular crowding agent containing both BSA and Ficoll 70 on
amyloid formation of HEL as a function of crowder concentration and composition
[17]. Both the mixed crowding agent and the protein crowding agent BSA (100
g/L) almost completely inhibited amyloid formation of lysozyme and stabilized
lysozyme activity on the investigated time scale. However, 100 g/L Ficoll 70
neither effectively impeded amyloid formation of lysozyme nor stabilized
lysozyme activity. By using ITC, we observed a weak, non-specific interaction
between BSA and nonnative lysozyme at pH 2.0. The ITC results are shown in Fig.
1(A,B). The best fit for the integrated heat data was obtained using a
three sequential-binding sites model, yielding the thermodynamic parameters for
the interaction between BSA and nonnative lysozyme.
Data
These results show that the binding of BSA to nonnative lysozyme is driven
entirely by large favorable enthalpy decreases but with unfavorable entropy
decreases for the first and the third sequential binding sites of nonnative
lysozyme. This implies that BSA may bind to lysozyme oligomers to prevent the
formation of prefibrillar lysozyme and bind with the protofibrils to retard
fibril elongation of lysozyme. Furthermore, we performed ITC experiments on the
binding of Ficoll 70 to nonnative lysozyme at pH 2.0 and found the calorimetric
data too small to fit any binding model [Fig. 1(C,D)]. No optimal
fit was found for Ficoll 70, indicating that it has no specific binding
affinity for nonnative lysozyme under such experimental conditions. Our ITC
analyses indicate that a mixture of 5 g/L BSA and 95 g/L Ficoll 70 inhibits
amyloid formation of lysozyme and maintains both lysozyme activity via mixed
macromolecular crowding and weak, nonspecific interactions between BSA and
nonnative lysozyme [17]. Our data demonstrated that BSA and Ficoll 70
cooperatively contribute to both the inhibitory effect and the stabilization
effect of the mixed crowding agent, suggesting that mixed macromolecular
crowding inside the cell may play a role in posttranslational quality control
mechanism [17].
a-synuclein is
the vital protein involved in neurodegenerative diseases, such as Parkinson’s
disease. Amyloid formation of recombinant human a-synuclein in vitro
can be accelerated by sodium dodecylsulfate (SDS). Ahmad et al employed ITC
and other biophysical methods to characterize the protein-detergent
interactions as a function of the concentration of SDS [18]. Their study showed
two types of ensembles of a-synuclein and SDS: the fibrillogenic ensembles formed with optimal
concentration of SDS around 0.5–0.75 mM, are characterized by enhanced accessible hydrophobic
surfaces and extended to partially helical conformation, while the less or
non-fibrillogenic ensembles formed above 2 mM SDS, are characterized by less
accessible hydrophobic surfaces and maximal helical content [18]. Their study
with the membrane-mimicking agent SDS should prove useful in understanding the
role of amphiphilic molecules in the fibrillogenicity of a-synuclein.
Amyloid fibrils share various common structural features, and their
presence can be detected by thioflavin T (ThT). Despite widespread use of ThT
for identifying amyloid fibrils, the mode for binding ThT to amyloid fibrils is
largely unknown. A detailed knowledge of the binding mode of ThT to amyloid fibrils
is essential for understanding the mechanism for protein misfolding. Groenning et
al examined the binding mode of ThT to insulin amyloid fibrils using ITC
and Scatchard analysis [19], and confirmed at least two binding site
populations. The binding site population with the strongest binding is
responsible for the characteristic ThT fluorescence. This binding has a
capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form.
The binding capacity is unaffected by pH, but the affinity is lowest at low pH.
Notably, the presence of a third binding process prior to the other processes
is suggested by ITC [19].
ITC Applications in
Protein-protein Interactions
Protein-protein interactions (PPI) play key roles in many essential biological
processes, such as the regulation of enzymatic activities, the assembly of
cellular components, and signal transduction [20]. ITC is the most quantitative
means available for measuring the thermodynamic properties of PPI and is
becoming a necessary tool for PPI complex structural studies [1–4,21–28].
Xanthine oxidase (XO) and copper, zinc superoxide dismutase
(Cu,Zn-SOD) are function-related proteins in vivo. We studied
thermodynamics of the interaction of bovine milk XO with bovine erythrocyte Cu,Zn-SOD
using ITC [21]. The binding of XO to Cu,Zn-SOD was driven by a large favorable
enthalpy decrease with a large unfavorable entropy reduction, and showed strong
entropy-enthalpy compensation and weak temperature-dependence of Gibbs free
energy change. An unexpected, large positive molar heat capacity change of the
binding, 3.02 kJ∙mol–1∙K–1, at all temperatures examined suggests that either hydrogen bond or
long-range electrostatic interaction is a major force for the binding. The
large unfavorable change in entropy suggests that long-range electrostatic
forces do not play an important role in the binding. These results indicate
that XO binds to Cu,Zn-SOD with high affinity and that hydrogen bond is a major
force for the binding [21].
Botulinum neurotoxins are produced by Clostridium botulinum
and cause the neuroparalytic syndrome of botulism. Jin et al reported
the structure of receptor-binding domain of botulinum neurotoxin serotype B and
the luminal domain of synaptotagmin II, which is the receptor of the neurotoxin
[22]. Their ITC data show that the carboxy-terminal domain of the heavy chain
of the neurotoxin binds tightly to the luminal domain of synaptotagmin II with
stoichiometry 1:1 and is endothermic and entropy driven. The heat capacity for the
interaction is approximately –326 cal∙mol–1∙K–1, which is consistent with a protein-protein interaction driven by
the hydrophobic effect. ITC titration at pH 5.7, mimicking the acidic endosomal
environment, produced no change on assembly thermodynamics, indicating that the
pH change associated with toxin internalization unlikely affects the binding of
the neurotoxin to its protein receptor [22].
The small ubiquitin-related modifier (SUMO) regulates a wide range
of cellular processes by post-translational modification with one or a chain of
SUMO molecules. Sumoylation is achieved by the sequential action of several
enzymes in which the E2, Ubc9, transfers SUMO from the E1 to the target mostly
with the help of an E3 enzyme. In this process, Ubc9 not only forms a thioester
bond with SUMO, but it also interacts with SUMO non-covalently [23]. Knipscheer
et al showed that this non-covalent interaction promotes the formation
of short SUMO chains on targets, such as Sp100 and HDAC4 [23]. ITC was used to
determine the affinity of the interaction between Ubc9 and SUMO1. For the
interaction between Ubc9 and SUMO1, a Kd of 8223
nM was determined. However, the heat exchange of the reaction for SUMO2 binding
was too small to be measured by ITC. Thus, in the two systems, the balance of
affinities is maintained in different ways, emphasizing that ubiquitin and SUMO
are analogous, but not identical. Nevertheless, the actual mechanism for chain
formation seems once again surprisingly similar between the ubiquitin and SUMO
pathways [23].
Protein phosphatase 2A (PP2A) is a major protein serine/threonine
phosphatase and is involved in many essential aspects of cellular physiology
[24]. The small T antigen (ST) of DNA tumor virus SV40 facilitates cellular
transformation by disrupting the functions of PP2A through a poorly defined
mechanism. Chen et al depicted the mechanism of ST regulation of PP2A
and quantitatively measured the binding affinities between ST and PP2A using
ITC [24]. They constructed a model that combined the affinity data and
structure features, and showed how ST may interfere with the normal functions
of PP2A. ST has a lower binding affinity than B56 for the PP2A core enzyme.
Consequently, ST does not efficiently displace B56 from PP2A holoenzymes in
vitro. Notably, ST inhibits PP2A phosphatase activity through its
N-terminal J domain. These findings suggest that ST may function mainly by
inhibiting the phosphatase activity of the PP2A core enzyme and, to a lesser
extent, by modulating assembly of the PP2A holoenzymes [24].
The recent finding of an interaction between calmodulin and the
tobacco mitogen-activated protein kinase (MAPK) phosphatase-1 has established
an important connection between Ca2+ signaling and the MAPK
cascade, two of the most important signaling pathways in plant cells. Rainaldi et
al characterized the binding of soybean calmodulin isoforms to synthetic
peptides derived from the calmodulin binding domain of the tobacco MAPK
phosphatase-1 [25]. Using ITC, they found that in the presence of Ca2+, the peptides bind first to the C-terminal lobe of calmodulin with
a nanomolar affinity, and at higher peptide concentrations, a second peptide
binds to the N-terminal domain with lower affinity. Thermodynamic analyses also
demonstrate that the formation of the peptide-bound complex with the Ca2+-loaded calmodulin is driven by favorable binding enthalpy due to a
combination of hydrophobic and electrostatic interactions [25].
Association of two proteins can be described as a two-step process,
with the formation of an encounter complex followed by desolvation and the
establishment of a tight complex. Kiel et al designed a set of mutants
of the Ras effector protein Ral guanine nucleotide dissociation stimulator
(RalGDS) with optimized electrostatic steering [26]. The results from ITC and
other biophysical methods showed that the fastest binding RalGDS mutant,
M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter than the wild
type. Upon further formation of the final complex, the increased Coulombic
interactions are probably counterbalanced by the cost of desolvation of
charges, keeping the dissociation rate constant almost unchanged. This
mechanism is also reflected by the mutual compensation of enthalpy and entropy
changes quantified by ITC. The binding constants of the faster binding RalGDS
mutants toward Ras are similar to those of Raf, the most prominent Ras
effector, suggesting that the design methodology may be used to switch between
signal transduction pathways [26].
ATP hydrolysis by the Hsp90 molecular chaperone requires a connected
set of conformational switches triggered by ATP binding to the N-terminal
domain in the Hsp90 dimer. Hsp90 mutants that influence these conformational
switches have strong effects on ATPase activity. ATPase activity is specifically
regulated by Hsp90 co-chaperones, which directly influence the conformational
switches. Using ITC and other biophysical methods, Siligardi et al
analyzed the effect of Hsp90 mutations on the binding and ATPase regulation by
the co-chaperones Aha1, Sti1, and Sba1 [27]. The ability of Sti1 to bind Hsp90
and arrest its ATPase activity was not affected by any of the mutants screened.
Sba1 bound in the presence of AMP-PNP to wild type and ATPase hyperactive
mutants with similar affinity, but it bound very weakly to hypoactive mutants
despite their wild-type ATP affinity. Unexpectedly, in all cases, Sba1 bound to
Hsp90 with a 1:2 molar stoichiometry. Analyses of complex formation with
co-chaperone mixtures have shown that Aha1 and p50cdc37 are
able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bind independently to Hsp90-AMP-PNP but not together. These data
have indicated that Sba1 and Aha1 regulate Hsp90 by influencing the
conformational state of the “ATP lid” and consequent N-terminal
dimerization, whereas Sti1 does not [27].
Elucidation of the roles of the hydrogen bonds involved in
antigen-antibody complementary association requires both structural and
thermodynamic information. Yokota et al examined the interaction between
HEL and its HyHEL-10 variable domain fragment (Fv) antibody [28]. They
constructed three antibody mutants and investigated the interactions between
the mutant Fvs and HEL. The results from ITC indicate that the mutations
significantly decreased the negative enthalpy change, despite some offset by a
favorable entropy change. X-ray crystallography demonstrate that the complexes
have nearly identical structures, including the positions of the interfacial
water molecules. Together, the ITC and X-ray crystallographic results indicate
that hydrogen bonding via interfacial water enthalpically contributes to the
Fv-HEL interaction despite the partial offset because of entropy loss,
suggesting that hydrogen bonding stiffens the antigen-antibody complex [28].
ITC Applications in
Protein-DNA/RNA Interactions
ITC has been used to study problems related to DNA and RNA
biochemistry. There have been several works in the area of protein-DNA/RNA
interactions [29–34]. Gel shift assays and size exclusion column studies are probably
the most common methods used to analyze DNA/RNA-protein interactions because of
their relative simplicity and their small sample preparations. However, ITC
provides some other advantages. The fast and automated machine can provide
direct thermodynamic information of enthalpy change, entropy change,
stoichiometry and binding constant.
Minetti et al employed ITC to investigate the binding of a
bifunctional repair enzyme, Escherichia coli formamidopyrimidine-glycosylase
(Fpg) to a series of 13-mer DNA duplexes as an initial step in defining the
thermodynamic profile of glycosylase-mediated DNA repair [29]. The ITC-binding
studies were carried out between 5 ºC and 15 ºC, and indicate that binding free
energies are relatively independent of temperature while the reaction enthalpy
and entropy are strongly temperature-dependent. The interaction is exclusively
an entropy-driven process that is characterized by a strongly unfavorable
binding enthalpy. The large negative heat capacity of the binding interaction
is consistent with Fpg complexation to the THF-containing duplexes involving
significant burial of non-polar surface areas. The structural and energetic
information from the thermodynamic investigations between Fpg and DNA duplexes
have led to a better understanding of the molecular forces that modulate lesion
recognition and repair [29].
Buczek et al measured the stoichiometry, enthalpy change, entropy
change and dissociation constant for binding telomere DNA fragments with the a protein
N-terminal domain at different temperatures and salt concentrations using ITC
[30]. Several telomere DNA fragments were synthesized, and thermodynamic
parameters of the binding to them of a subunit of the telomere end-binding protein
were reported. Their results show that each fragment forms a monovalent protein
complex with the protein except for the fragment d(T4G4T4G4), which has two
tandemly repeated d(TTTTTGGGG) telomere motifs with a high-affinity binding
site and a low-affinity binding site. The relative contributions of entropy
change and enthalpy change for binding reactions are DNA length-dependent, as
is negative heat capacity change. These results are important for understanding
early intermediate and subsequent stages in the assembly of the full telomere
nucleoprotein complex and how binding events can prepare the telomere DNA for
extension by telomerase, a critical event in telomere biology [30].
Using ITC, Ziegler et al observed that HIV-1 Tat (47–57) [31], a
cell-penetrating peptide (CPP), has a high affinity for double-stranded salmon
sperm DNA, as characterized by a dissociation constant of 126 nM. The observed
dissociation constant for binding of HIV-1 Tat-PTD to DNA was only slightly
higher than that for the specific interaction of the full-length HIV-1 Tat to
TAR, which confers stability to the uptake complexes of extracellular DNA and
CPPs, and also points to the potential interference of the CPP with
intracellular DNA as well as the competitive release of the cargo after
cellular uptake. The binding is exothermic, and the dissociation constant and
reaction enthalpy decrease further at higher temperatures. The high value of
entropy likely reflects the release of hydration water and counter-ions during
polyelectrolyte binding and condensation, which light-scattering data also
support. Both favorable negative enthalpy and favorable positive entropy drive
the binding reaction, and they are even more favorable at higher temperatures
[31].
ITC analyses of the binding of human cytomegalovirus DNA polymerase
UL44 to several different double-stranded DNA have been characterized by
Loregian et al [32]. UL44 binds to DNA as a dimer, and that binding is
entropically driven, while the dependence of binding on DNA length exists,
which are consistent with the results of electrophoretic mobility shift assays.
They have also suggested a minimum DNA length for UL44 interactions. The
thermodynamic investigation has furthered understanding of how the human
cytomegalovirus DNA polymerase accessory protein interacts with DNA and has
also provided some insight into its mechanism of processivity [32].
Recht et al determined thermodynamics of the cooperativity in
the assembly of the central domain from the 30S rRNA subunit of A. aeolicus
by ITC [33]. They observed that there is cooperativity in the binding of S15,
S6 and S18, but binding of S8 and S11 is independent of all other proteins from
the enthalpy of each binding event. These results suggest that
interdependencies of protein binding in the assembly of the A. aeolicus
central domain are similar, but not identical, to those observed in the E.
coli assembly map [33].
Volpon et al carried out ITC experiments to detect a short
single-stranded RNA binding with TcUBP1 [34], a trypanosome cytoplasmic
RNA-binding protein containing a single and conserved RNA-recognition motif
domain involved in selective destabilization of U-rich mRNAs. The RNA binding
reaction was driven by a large negative enthalpy change, suggesting the
formation of hydrogen bonds, van der Waals contacts, and/or electrostatic
interactions. Given the polar and charged nature of RNA, it is likely that
hydrogen-bond/electrostatic interactions contribute significantly to the
binding reaction. A large negative entropy change accompanied the binding
events, indicating an increase in order during the RNA binding due to the
reduction in the translational and rotational degrees of freedom of the RNA and
the protein side chains engaged in complex formation [34].
ITC Applications in
Protein-small Molecule Interactions
An understanding of the molecular basis of protein-small molecule
interactions is crucial to attempts to design novel drug technologies. The
thermodynamic information of the interactions of protein-small molecule
obtained from ITC facilitates the understanding of the binding modes and is
helpful to the development of novel drugs for some serious diseases [35–41].
Ferulic acid (FA) is one of the most effective components of the
traditional Chinese medicine Angelica, and cytochrome c plays a
vital role in apoptosis. We reported the application of ITC and several
biophysical methods to investigate the mechanism for the interaction between
bovine heart cytochrome c and FA as well as the effect of the binding on
native state stability of the protein at physiological pH [35]. ITC studies
together with fluorescence spectroscopic measurements indicate that FA binds to
cytochrome c with moderate affinity and quenches the intrinsic
fluorescence of the protein in a static way. The interaction of cytochrome c
with FA is driven by a moderately favorable entropy increase in combination
with a less favorable enthalpy decrease for the first binding site of the
protein. The melting temperature of cytochrome c in the presence of FA
measured by differential scanning calorimetry and circular dichroism increases
4 ºC and 5 ºC respectively, compared with that in the absence of FA. Taken
together, these results indicate that FA binds to and stabilizes cytochrome c
at physiological pH. Furthermore, binding of FA to cytochrome c inhibits
cytochrome c-induced apoptosis of human hepatoma cell line SMMC-7721.
Our data provide insight into the mechanism of drug-protein interactions and
will be helpful in understanding the mechanism for FA-inhibited and cytochrome c-induced
apoptosis [35].
Bicyclomycin is the only natural product inhibitor with weak binding
affinity of the transcription termination factor rho, which is a hexameric
helicase that terminates nascent RNA transcripts utilizing ATP hydrolysis and
is an essential protein for many bacteria. Brogan et al determined
the information concerning a bicyclomycin analogue-rho interaction using ITC
[36]. Their study found that a designed bicyclomycin ligand,
5a-(3-formyl-phenylsulfanyl)-dihydrobicyclomycin, inhibits rho an order of
magnitude more efficiently than bicyclomycin [36].
Organisms rely heavily on protein phosphorylation to transduce
intracellular signals. The phosphorylation of a protein often induces
conformational changes, which are responsible for triggering downstream
cellular events. Engel et al developed some specific, low molecular
weight compounds that target the hydrophobic motif/PIF-pocket and have the
ability to allosterically activate phosphoinositide-dependent protein kinase
1 (PDK1) by modulating the phosphorylation-dependent conformational transition
[37]. The interaction of compound 1 with PDK1 was studied using ITC, and the
experiments indicate that compound 1 binds to PDK1 CD with a 1:1 stoichiometry
and a binding affinity in the micromolar range. These results raise the
possibility of developing drugs that target the AGC kinases via a novel mode of
action and may inspire future rational development of compounds with the
ability to modulate phosphorylation-dependent conformational transitions in
other proteins [37].
An efficient research strategy integrating empirically guided,
structure-based modeling and chemoinformatics was used to discover potent small
molecule inhibitors of the botulinum neurotoxin serotype A (botulinum A) light
chain. Using ITC, Burnett et al studied the interaction between a small
molecule NSC 240898 and the botulinum A light chain [38]. The inhibitor
interaction with the botulinum A light chain is a low affinity binding event
with a 1:1 stoichiometry. Furthermore, the interaction is largely
entropy-driven, and the enthalpic component is relatively low. The substantial
entropic contribution to the binding event suggests a burial of hydrophobic
surfaces and the release of solvent [38].
The E. coli isocitrate lyase regulator (IclR) regulates the
expression of the glyoxylate bypass operon. IclR comprises a DNA binding domain
that interacts with the operator sequence and a C-terminal domain that binds a
hitherto unknown small molecule. Glyoxylate and pyruvate, identified by Lorca et
al, bind to the C-IclR domain, as defined by ITC [39]. The titration of
C-IclR with each compound followed an exothermal heat change profile, giving
rise to a sigmoidal binding curve with glyoxylate or hyperbolic with pyruvate.
The stoichiometry of the reaction, 0.5, was consistent with the binding of one
ligand molecule per IclR dimer. In accordance with other results, the C-IclR
dissociation constant for glyoxylate was significantly lower than that for
pyruvate. In general, their strategy of combining chemical screens with
functional assays and structural studies has uncovered two small molecules with
antagonistic effects that regulate the IclR-dependent transcription of the aceBAK
operon [39].
Organisms commit a considerable amount of genetic and metabolic
resources to managing metal ions. This involves proteins dedicated to the
uptake, transport, storage and export of essential metal as well as their
delivery to proteins and enzymes requiring one or more metals for their
stability and/or catalytic activity [40]. A review by Wilcox highlighted many
of the recent studies of metal ions binding to proteins that have used ITC to
quantify the thermodynamics of metal-protein interactions [40].
The cellular prion protein is known to be a copper-binding protein.
Thompsett et al used two techniques, ITC and competitive metal capture
analysis, to determine the affinity of copper for wild-type mouse PrP and a
series of mutants [41]. High affinity copper binding by wild-type PrP was
confirmed by independent techniques, which indicated the presence of specific
tight copper binding sites up to femtomolar affinity. Altogether, four high
affinity binding sites of between femto- and nanomolar affinities were located
within the octameric repeat region of the protein at physiological pH. A fifth
copper binding site of lower affinity than those of the octameric repeat region
was detected in full-length protein. Binding to this site is modulated by the
histidine at residue 111. Removal of the octameric repeats led to the
enhancement of affinity of this fifth site and a second binding site outside of
the repeat region undetected in the wild-type protein. High affinity copper
binding allows PrP to compete effectively for copper in the extracellular
milieu. The copper binding affinities of PrP were compared with those of
proteins of known function, and they are of magnitudes compatible with an
extracellular copper buffer or an enzymatic function, such as superoxide
dismutase-like activity [41].
Reverse Titration of ITC
The injected reactant located in the syringe is referred to as
“ligand”. Usually small molecules should be placed in the syringe,
and the targeted protein should be placed in the cell. Sometimes reverse
titrations (i.e., reversing the role of macromolecule and ligand) are conducted
to check the stoichiometry or the suitability of the binding model [42].
In the present study, I have used reverse titration of ITC to
measure the binding affinity of oleic acid to Ca2+-depleted
bovine a-lactalbumin (apo-BLA). A reverse titration of 287 mM apo-BLA into
36 mM
oleic acid using 2810-ml injections was performed because of the insolubility of oleic
acid, and the ITC results are shown in Fig. 2(A,B). The
best fit for the integrated heat data was obtained using a three
sequential-binding sites model, yielding the thermodynamic parameters for the
interaction between apo-BLA and oleic acid.
Data
Our results show that the binding of oleic acid to apo-BLA is driven
entirely by large favorable entropy increases but with unfavorable enthalpy
increases for the first and the third sequential-binding sites of nonnative
lysozyme. The oscillating calorimetric signal indicates the formation of the
aggregates of apo-BLA induced by oleic acid.
To characterize driving forces and driven processes in the formation
of a large interface, wrapped protein-DNA complex analogous to the nucleosome,
Vander Meulen et al investigated the thermodynamics of binding the 34 bp
H DNA sequence to the E. coli DNA-remodeling protein integration host
factor (IHF) [43]. Thermodynamic parameters for integration host factor-H DNA
interactions were determined by ITC from forward and reverse titrations. Both
the binding constant and the binding enthalpy depend strongly on salt
concentration and anion identity. Formation of the wrapped complex is enthalpy
driven, especially at a low concentration of salt [43].
The HIV-1 nucleocapsid (NC) protein is a small, basic protein
containing two retroviral zinc fingers. NC binds with high-affinity to the
repeating sequence d(TG)n. The interactions between NC and (TG)4 have been characterized by ITC [44]. The forward titration curve
reaches saturation at a molar ratio of approximately 1.0. A reverse titration
in which NC was titrated into a solution of 10 mM (TG)4 was
also performed, but no clear saturation was observed. The dependence of the
total heat released upon the direction of the titration also underscores the
complexity of the interactions between NC and (TG)4. The
role of electrostatic interactions in the binding was probed, both by repeating
the titration of NC into (TG)4 in varying concentrations of
NaCl and by using the “N-term” mutant protein. The amount of heat
released was drastically reduced in both of these experiments, suggesting that
that Coulombic attractions play a major role in the interactions between NC and
(TG)4 [44].
Displacement Method of ITC
An important goal in drug development is to engineer inhibitors and
ligands that have high binding affinities for their target molecules. In
optimizing these interactions, the precise determination of the binding
affinity becomes progressively difficult once it approaches and surpasses the
nanomolar level. ITC can be used to determine the complete binding
thermodynamics of a ligand down to the picomolar range by using an experimental
mode called displacement titration, a new and important progress in ITC [42].
In the recent years, this displacement method has been applied successfully in
calorimetry when dealing with very high or very low affinity systems [42,45].
This method is based on the fact that the binding properties of a ligand are
altered when another competing ligand is present.
In a displacement titration, the association constant of a
high-affinity ligand that cannot be measured directly is artificially lowered
to a measurable level by premixing the protein with a weaker competitive
ligand. To perform this protocol, three titrations must be carried out: a
direct titration of the high-affinity ligand to the target protein, a direct
titration of the weak ligand to the target protein and a displacement titration
of the high-affinity ligand to the weak ligand-target protein complex [42]. In
a displacement titration, the weak competitive ligand must be present in the
calorimetric cell at a concentration sufficient to reduce the affinity of the
high-affinity ligand to measurable levels (Ka109 M–1). The apparent binding affinity
of the high-affinity ligand, , is reduced by a factor, RF, dependent on the
binding affinity, KaX, and the concentration of the
weak ligand [42]:
Eq.
where [X] is the concentration of the free weak ligand X, which is unknown.
For practical purposes, the total concentration of a weak ligand required to
achieve a predetermined reduction factor is approximately [42]:
Eq.
where [X]T and [M]T are the
total weak ligand and total macromolecule concentration in the calorimetric
cell. The displacement method of ITC has been used successfully to determine
the binding constant of a high-affinity HIV-1 protease inhibitor using
acetyl-pepstatin as the weak inhibitor [42].
If we are interested in characterizing a very low affinity ligand,
then a moderate affinity ligand is used as competing ligand and a thermodynamic
principle similar to the above is used [45]. Three titrations are performed: a
direct titration of moderate affinity ligand into the protein solution, from
which the both the binding affinity and the binding enthalpy can be obtained; a
direct titration of low affinity ligand into the protein solution, from which
neither the binding affinity nor the binding enthalpy can be reliably obtained;
and displacement titration of moderate affinity ligand A into a solution of
macromolecule and ligand B [45].
In the work of Andújar-Sánchez et al [46], the binding
constants of angiotensin-converting enzyme inhibitors were determined by a
displacement method of ITC. Somatic angiotensin I-converting enzyme (s-ACE)
plays a central role in blood pressure regulation and has been the target of
most antihypertensive drugs. Direct ITC titrations were made to determine
binding enthalpy and binding constants for L-Asp-L-Phe. Binding constants for
the strong inhibitors, captopril, lisinopril and enalaprilat, were measured by
the displacement method. For each displacement experiment, a solution of s-ACE
was first titrated until saturation with the weak inhibitor L-Asp-L-Phe. Then,
the injection syringe was cleaned and refilled with a solution of the strong
inhibitor to perform a second titration. The relative potency of the inhibitors
was determined to be enalaprilat>lisinopril>captopril.
Andújar-Sánchez et al analyzed the thermodynamic behavior of the binding
process using the new structural information provided by the ACE structures, as
well as the conformational changes that occurred upon binding [46].
Conclusions
The ITC method is gaining wider usage with respect to investigating protein
interactions in signal transduction and deeper usage with respect to
investigating protein folding and misfolding. The proliferation of this method
in academic and industrial laboratories has produced a lot of new reports of
interesting applications, new systems studied and advances in data analyses.
Here, the new application of ITC in protein folding and misfolding, as well as
its traditional application in protein interactions is reviewed. From analyzing
the experimental data, scientists have gained a better understanding of the
relationships between the ITC data and structural details. Methods for
analyzing ITC still need to be further developed to ensure the effectiveness of
ITC results. Combining X-ray crystallography and nuclear magnetic resonance
spectroscopy with ITC may be one method that will help provide greater
understanding of the complexities of protein folding and protein interactions.
References
1 Cliff MJ, Gutierrez A, Ladbury JE. A survey
of the year 2003 literature on applications of isothermal titration
calorimetry. J Mol Recognit 2004, 17: 513–523
2 Ababou A, Ladbury JE. Survey of the year
2004: literature on applications of isothermal titration calorimetry. J Mol
Recognit 2006, 19: 79–89
3 Ababou A, Ladbury JE. A survey of the year
2005 literature on applications of isothermal titration calorimetry. J Mol
Recognit 2007, 20: 4–14
4 Okhrimenkoa O, Jelesarov I. A survey of the
year 2006 literature on applications of isothermal titration calorimetry. J Mol
Recognit 2008, 21: 1–19
5 Baker BM, Murphy KP. Evaluation of linked
protonation effects in protein binding reactions using isothermal titration
calorimetry. Biophys J 1996, 71: 2049–2055
6 Connelly PR, Varadarajan R, Sturtevant JM,
Richards FM. Thermodynamics of protein-peptide interactions in the ribonuclease
S system studied by titration calorimetry. Biochemistry 1990, 29: 6108–6114
7 Spolar RS, Record MT Jr. Coupling of local
folding to site-specific binding of proteins to DNA. Science 1994, 263: 777–784
8 Anfinsen CB. Principles that govern the
folding of protein chains. Science 1973, 181: 223–230
9 Liang Y, Du F, Sanglier S, Zhou BR, Xia Y,
Van Dorsselaer A, Maechling C et al. Unfolding of rabbit muscle creatine
kinase induced by acid. A study using electrospray ionization mass
spectrometry, isothermal titration calorimetry, and fluorescence spectroscopy.
J Biol Chem 2003, 278: 30098–30105
10 Fan YX, Zhou JM, Kihara H, Tsou CL. Unfolding
and refolding of dimeric creatine kinase equilibrium and kinetic studies.
Protein Sci 1998, 7: 2631–2641
11 Nakamura S, Kidokoro S. Direct observation of
the enthalpy change accompanying the native to molten-globule transition of
cytochrome c by using isothermal acid-titration calorimetry. Biophys Chem 2005,
113: 161–168
12 Luke K, Wittung-Stafshede P. Folding and
assembly pathways of co-chaperonin proteins 10: Origin of bacterial
thermostability. Arch Biochem Biophys 2006, 456: 8–18
13 Yang F Jr, Zhang M, Zhou BR, Chen J, Liang Y. Oleic
acid inhibits amyloid formation of the intermediate of a-lactalbumin at
moderately acidic pH. J Mol Biol 2006, 362: 821–834
14 Kardos J, Yamamoto K, Hasegawa K, Naiki H,
Goto Y. Direct measurement of the thermodynamic parameters of amyloid formation
by isothermal titration calorimetry. J Biol Chem 2004, 279: 55308–55314
15 Zhou BR, Liang Y, Du F, Zhou Z, Chen J. Mixed
macromolecular crowding accelerates the oxidative refolding of reduced,
denatured lysozyme: implications for protein folding in intracellular
environments. J Biol Chem 2004, 279: 55109–55116
16 Du F, Zhou Z, Mo ZY, Shi JZ, Chen J, Liang Y.
Mixed macromolecular crowding accelerates the refolding of rabbit muscle
creatine kinase: implications for protein folding in physiological environments.
J Mol Biol 2006, 364: 469–482
17 Zhou BR, Zhou Z, Hu QL, Chen J, Liang Y. Mixed
macromolecular crowding inhibits amyloid formation of hen egg white lysozyme.
Biochim Biophys Acta 2008, 1784: 472–480
18 Ahmad MF, Ramakrishna T, Raman B, Rao ChM.
Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human a-synuclein. J Mol
Biol 2006, 364: 1061–1072
19 Groenning M, Norrman M, Flink JM, van de Weert
M, Bukrinsky JT, Schluckebier G, Frokjaer S. Binding mode of thioflavin T in
insulin amyloid fibrils. J Struct Biol 2007, 159: 483–497
20 Zhou YL, Liao JM, Chen J, Liang Y.
Macromolecular crowding enhances the binding of superoxide dismutase to
xanthine oxidase: implications for protein-protein interactions in
intracellular environments. Int J Biochem Cell Biol 2006, 38: 1986–1994
21 Zhou YL, Liao JM, Chen J, Liang Y.
Thermodynamics of the interaction of xanthine oxidase with superoxide dismutase
by isothermal titration calorimetry and fluorescence spectroscopy. Thermochim
Acta 2005, 426: 173–178
22 Jin R, Rummel A, Binz T, Brunger AT. Botulinum
neurotoxin B recognizes its protein receptor with high affinity and
specificity. Nature 2006, 444: 1092–1095
23 Knipscheer P, van Dijk WJ, Olsen JV, Mann M,
Sixma TK. Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain
formation. EMBO J 2007, 26: 2797–2807
24 Chen Y, Xu Y, Bao Q, Xing Y, Li Z, Lin Z,
Stock JB et al. Structural and biochemical insights into the regulation
of protein phosphatase 2A by small t antigen of SV40. Nat Struct Mol Biol 2007,
14: 527–534
25 Rainaldi M, Yamniuk AP, Murase T, Vogel HJ.
Calcium-dependent and -independent binding of soybean calmodulin isoforms to
the calmodulin binding domain of tobacco MAPK phosphatase-1. J Biol Chem 2007,
282: 6031–6042
26 Kiel C, Selzer T, Shaul Y, Schreiber G,
Herrmann C. Electrostatically optimized Ras-binding Ral guanine dissociation
stimulator mutants increase the rate of association by stabilizing the
encounter complex. Proc Natl Acad Sci U S A 2004, 101: 9223–9228
27 Siligardi G, Hu B, Panaretou B, Piper PW,
Pearl LH, Prodromou C. Co-chaperone regulation of conformational switching in
the Hsp90 ATPase cycle. J Biol Chem 2004, 279: 51989–51998
28 Yokota A, Tsumoto K, Shiroishi M, Kondo H,
Kumagai I. The role of hydrogen bonding via interfacial water molecules in
antigen-antibody complexation. The HyHEL-10-HEL interaction. J Biol Chem 2003,
278: 5410–5418
29 Minetti CA, Remeta DP, Zharkov DO, Plum GE,
Johnson F, Grollman AP, Breslauer KJ. Characterization of
formamidopyrimidine-glycosylase(Fpg) interactions with damaged DNA duplexes. J
Mol Biol 2003, 328: 1047–1060
30 Buczek P, Horvath MP. Thermodynamic
characterization of binding Oxytricha nova single strand telomere DNA
with the alpha protein N-terminal domain. J Mol Biol 2006, 359: 1217–1234
31 Ziegler A, Seelig J. High affinity of the
cell-penetrating peptide HIV-1 Tat-PTD for DNA. Biochemistry 2007, 46: 8138–8145
32 Loregian A, Sinigalia E, Mercorelli B, Palù G,
Coen DM. Binding parameters and thermodynamics of the interaction of the human
cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications
for the processivity mechanism. Nucleic Acids Res 2007, 35: 4779–4791
33 Recht MI, Williamson JR. RNA tertiary
structure and cooperative assembly of a large ribonucleoprotein complex. J Mol
Biol 2004, 344: 395–407
34 Volpon L, D’Orso I, Young CR, Frasch AC,
Gehring K. NMR structural study of TcUBP1, a single RRM domain protein from Trypanosoma
cruzi: contribution of a b hairpin to RNA binding.
Biochemistry 2005, 44: 3708–3717
35 Yang F, Zhou BR, Zhang P, Zhao YF, Chen J,
Liang Y. Binding of ferulic acid to cytochrome c enhances stability of the
protein at physiological pH and inhibits cytochrome c-induced apoptosis. Chem
Biol Interact, 2007, 170: 231–243
36 Brogan AP, Widger WR, Bensadek D, Riba-Garcia
I, Gaskell SJ, Kohn H. Development of a technique to determine bicyclomycin-rho
binding and stoichiometry by isothermal titration calorimetry and mass
spectrometry. J Am Chem Soc 2005, 127: 2741–2751
37 Engel M, Hindie V, Lopez-Garcia LA, Stroba A,
Schaeffer F, Adrian I, Imig J et al. Allosteric activation of the
protein kinase PDK1 with low molecular weight compounds. EMBO J 2006, 25: 5469–5480
38 Burnett JC, Ruthel G, Stegmann CM, Panchal
RG., Nguyen TL, Hermone AR, Stafford RG et al. Inhibition of
metalloprotease botulinum serotype A from a pseudo-peptide binding mode to a
small molecule that is active in primary neurons. J Biol Chem 2007, 282: 5004–5014
39 Lorca GL, Ezersky A, Lunin VV, Walker JR,
Altamentova S, Evdokimova E, Vedadi M et al. Glyoxylate and pyruvate are
antagonistic effectors of the Escherichia coli IclR transcriptional
regulator. J Biol Chem 2007, 282: 16476–16491
40 Wilcox DE. Isothermal titration calorimetry of
metal ions binding to proteins: an overview of recent studies. Inorganica Chim
Acta 2008, 361: 857–867
41 Thompsett AR, Abdelraheim SR, Daniels M, Brown
DR. High affinity binding between copper and full-length prion protein
identified by two different techniques. J Biol Chem 2005, 280: 42750–42758
42 Velazquez-Campoy A, Freire E. Isothermal
titration calorimetry to determine association constants for high-affinity
ligands. Nat Protoc 2006, 1:186–191
43 Vander Meulen KA, Saecker RM, Record MT Jr.
Formation of a wrapped DNA-protein interface: experimental characterization and
analysis of the large contributions of ions and water to the thermodynamics of
binding IHF to H’ DNA. J Mol Biol 2008, 377: 9–27
44 Fisher RJ, Fivash MJ, Stephen AG, Hagan NA,
Shenoy SR, Medaglia MV, Smith LR et al. Complex interactions of HIV-1
nucleocapsid protein with oligonucleotides. Nucleic Acids Res 2006, 34: 472–484
45 Velázquez Campoy A, Freire E. ITC in the
post-genomic era…? Priceless. Biophys Chem 2005, 115: 115–124
46 Andújar-Sánchez M, Jara-Pérez V,
Cámara-Artigas A. Thermodynamic determination of the binding constants of
angiotensin-converting enzyme inhibitors by a displacement method. FEBS Lett
2007, 581: 3449–3454