advances-in-protein-chemistry

The total heat content Q of the solution in calorimeter cell (of volume V) is given by:
Q=nFM t
∆HV (25)
where M t
is total macromolecule concentration in calorimetric cell, F is the fraction of sites on M occupied by L and n
is the number of sites [112,116]. Substituting for the value of M t
from equation 21 in equation 22 leads to estimation of
dissociation constant (K d
=1/K A
). (More elaborate treatment of equations can be looked in [112,114,116]. Final data is
plotted as differential heat evolved for each injection of an aliquot of ligand L into sample in measurement cell versus the
molar ratio L t
/M t
. Data fitting is performed using non-linear least squares (NLLS) method and initial guesses are made
for K A
, ΔH and n and are varied till best fit of data is obtained [116]. Thus in ITC in a single experiment the values of the
dissociation constant (K d
), the stoichiometry (n) and the enthalpy of binding (ΔH) are determined. The free energy and
entropy of binding are determined from:
∆G=-RtlnKA=∆H-T∆S (26)
where ΔG is Gibbs free energy of binding, R is the universal gas constant, T is the temperature in Kelvin, K A
is the association
constant, ΔH is enthalpy change (heat) and ΔS is the entropy change on binding.
As ITC is a label-free method that can measure any reaction that results in heat change, it has found wide application in
protein characterization like studying protein-DNA [112,114,115], protein-protein [117,118] and protein-small molecule
interactions [113,117-120]. ITC happens to be the only technique that can directly measure energetics including Gibbs
free energy, enthalpy, entropy and heat capacity changes. Consequently in addition to binding thermodynamics ITC can
also be used to study catalytic reactions, conformational rearrangements and molecular dissociations [117,121,122]. An
important role for ITC has come up in structure-based drug design and other drug discovery strategies [117-119]. Free
energy changes associated with molecular interactions are a result of characteristics of interaction, like burial of apolar
surfaces or decrease in exposure of nonpolar surface or may be accompanied by displacement of buried water molecules.
Information from insights into thermodynamics of interaction of drug molecules with proteins has been helpful in aiding
the design of inhibitors and drug like molecules.
Differential scanning calorimetry
While ITC is conducted in presence of constant temperature, Differential Scanning Calorimetry (DSC) measures heat
capacity (C p
) and enthalpy of thermally induced transitions in particular conformational transitions in biological molecules
[113,114,123-125]. To put it simply, DSC provides a complete thermodynamic profile for unfolding energetics of the system
[113]. A typical DSC experiment involves monitoring difference of temperature in two cells; a reference cell that contains
reaction buffer and the sample cell that contains the reaction mixture which can be protein alone or mixture of protein and
an interacting molecule [114,123,126]. Both cells are electrically heated at a known constant temperature. A temperature
induced transition typically endothermic in nature is created in both cells but temperature of the sample cell lags behind
that of reference cell. An amount of compensatory electric power driven by a feedback mechanism is used to maintain the
two cells at same temperature. This amount of power (units Js -1 ) at temperature T divided by the heating rate (units Ks -1 ) is
solv
solv
the apparent difference in heat capacity between sample cell (C
p
) and reference cell (C
p
) , ΔC p
(units JK -1 ). This power
difference can be expressed as:
sol solv
∆ C = C −C (27)
p p p
Thermal transition curves in terms of heat capacity are inadequate because they are normalized to mass of substrate
(C p
can have units JK -1 or JK -1 g -1 ). A more meaningful comparison of the thermal transition curves of different substrates
or same substrate in different conditions (like ligand binding) can be achieved by normalizing concentrations according to
molarity. This new entity is called molar heat capacity (units JK-1mol-1) and is obtained by multiplying C p
by the molecular
weight (g mol -1 ).
For simple proteins a single heat absorption peak called ‘thermogram’ is usually observed in the scan. Applying correction
for instrument baseline and transition baseline and normalizing concentration, a direct calorimetric measurement of
enthalpy (ΔH) and melting temperature (T m
) can be obtained by integration of the thermogram with respect to temperature.
T
∆ =∫
H Cp.
dT (28)
T0
In protein characterization DSC has found application in studying equilibrium thermodynamic stability and folding
mechanism [123-126]. Protein stability through DSC is observed by estimating heat capacity increment that accompanies
protein denaturation or in simpler terms heat capacity of denaturation. It is positive because heat capacity of unfolded
protein is greater than that of native protein [123,124]. Melting temperature (T m
) is a good indicator of protein thermo
stability and generally higher the T m
, the more thermodynamically stable protein is. In addition to protein stability studies as
mentioned above and in previous section, DSC along with ITC form a formidable combination that have a role in studying
protein interactions [113-117,124-129]. The role of DSC has become all the more important when studying protein-DNA
interactions [114,130,131]. Since both DNA and protein structure significantly depend upon temperature the combined use
of ITC with DSC is made with data obtained from DSC experiments used for correcting ITC data.
Summary
Major aim of protein characterization is to arrive at meaningful and empirical information about these biomolecules. This information is a
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