1.3 A Brief
Introduction to the
Techniques Used
The aim of this chapter is not to give an extensive theoretical
introduction to each technique, since a wealth of such information
is available elsewhere in this volume. However, for the sake of those
readers who may be unfamiliar with a particular technique and have
not yet consulted other chapters, a short introduction is given
below.
Thermal shift assay: This assay exploits the stabilization of
proteins against thermal denaturation by mass action
(Le Chatelier’s principle) when small-molecule ligands bind selectively to the folded state and not to the unfolded state [9]. This
causes a shift in the midpoint of the typically sigmoidal unfolding
transition, which can be monitored by a variety of biophysical
methods including differential scanning fluorimetry (DSF) using
dyes that change their signal upon interacting with unfolded protein or using changes in intrinsic protein fluorescence upon unfolding. It is a high-throughput, low-consumption assay that can be
used to screen for the presence of binding, to identify promising
candidates for analysis by other methods, and to rank molecules
that bind to a particular site in order of affinity [10, 11]. For further
details about DSF and thermal shift assays, readers should consult
Chapter 8.
Fluorescence intensity: This technique measures changes in
intrinsic protein fluorescence intensity upon ligand binding, resulting from changes in the electronic and solvent environment of
tryptophan and tyrosine residues in the bound and unbound states
of the protein. This assay uses unadulterated native protein and is
simple to perform for small-molecule ligands that do not absorb
strongly or fluoresce in the same wavelength range as proteins.
Microscale thermophoresis (MST): This technique measures
changes in partitioning of macromolecules across a local temperature gradient (thermophoresis) upon ligand binding, resulting
from changes in surface area, ionic-shielding entropy, and solvation
entropy [12]. Changes in distribution of one binding partner
(which must be fluorescent, see Note 2) due to thermophoresis at
different concentrations of ligand are quantified via changes in
fluorescence intensity in a region that is transiently heated by an
infrared laser. This is a low-consumption assay and applicable to
many experimental contexts [13]. For further details about MST,
readers should consult Chapter 6.
Isothermal titration calorimetry (ITC): This technique measures the heat absorbed or evolved upon titration of ligand solution
into protein solution, using power compensation calorimetry. The
characteristic pattern of heat transfer during the titration depends
upon the enthalpy change for binding and the dissociation constant
for the interaction [14, 15]. Although requiring relatively large
amounts of material, this technique is close to universal in applicability to biomolecular binding events and can afford a more complete picture of the thermodynamics of binding than other
Interactions by Multiple Methods
49
Introduction to the
Techniques Used
The aim of this chapter is not to give an extensive theoretical
introduction to each technique, since a wealth of such information
is available elsewhere in this volume. However, for the sake of those
readers who may be unfamiliar with a particular technique and have
not yet consulted other chapters, a short introduction is given
below.
Thermal shift assay: This assay exploits the stabilization of
proteins against thermal denaturation by mass action
(Le Chatelier’s principle) when small-molecule ligands bind selectively to the folded state and not to the unfolded state [9]. This
causes a shift in the midpoint of the typically sigmoidal unfolding
transition, which can be monitored by a variety of biophysical
methods including differential scanning fluorimetry (DSF) using
dyes that change their signal upon interacting with unfolded protein or using changes in intrinsic protein fluorescence upon unfolding. It is a high-throughput, low-consumption assay that can be
used to screen for the presence of binding, to identify promising
candidates for analysis by other methods, and to rank molecules
that bind to a particular site in order of affinity [10, 11]. For further
details about DSF and thermal shift assays, readers should consult
Chapter 8.
Fluorescence intensity: This technique measures changes in
intrinsic protein fluorescence intensity upon ligand binding, resulting from changes in the electronic and solvent environment of
tryptophan and tyrosine residues in the bound and unbound states
of the protein. This assay uses unadulterated native protein and is
simple to perform for small-molecule ligands that do not absorb
strongly or fluoresce in the same wavelength range as proteins.
Microscale thermophoresis (MST): This technique measures
changes in partitioning of macromolecules across a local temperature gradient (thermophoresis) upon ligand binding, resulting
from changes in surface area, ionic-shielding entropy, and solvation
entropy [12]. Changes in distribution of one binding partner
(which must be fluorescent, see Note 2) due to thermophoresis at
different concentrations of ligand are quantified via changes in
fluorescence intensity in a region that is transiently heated by an
infrared laser. This is a low-consumption assay and applicable to
many experimental contexts [13]. For further details about MST,
readers should consult Chapter 6.
Isothermal titration calorimetry (ITC): This technique measures the heat absorbed or evolved upon titration of ligand solution
into protein solution, using power compensation calorimetry. The
characteristic pattern of heat transfer during the titration depends
upon the enthalpy change for binding and the dissociation constant
for the interaction [14, 15]. Although requiring relatively large
amounts of material, this technique is close to universal in applicability to biomolecular binding events and can afford a more complete picture of the thermodynamics of binding than other
Interactions by Multiple Methods
49