The simplest possible binding scheme
for a Ligand-Acceptor Interaction
Dissociation
6A@6B@
KD =
6A.B@
Association
6A.B@
1
KA = K =
D
6A@6B@
• To
determine the equilibrium constant you generally
titrate the Acceptor (A) with the ligand (B) and observe
the system using an appropriate experimental technique.
• That
technique must either deliver the concentration of
the unbound ligand or acceptor ([A] or [B]) or detect a
change in some physiochemical property of the ligand or
acceptor associated with binding.
Thursday, 28 October 2010
Some Physical Techniques for studying
Protein-ligand interactions
Spectroscopic Techniques
•UV absorbance spectroscopy ✔
•Fluorescence Spectroscopy ✔
•Circular Dichroism (CD) spectroscopy
•Fluorescence Anisotropy
•IR and Raman Spectroscopy
•NMR spectroscopy ✔
•EPR spectroscopy
✔
Calorimetric Techniques
•Isothermal Titration Calorimetry (ITC) ✔
•Differential Scanning Calorimetry (DSC)
Techniques Based on Migration and Separation
•Electrophoretic techniques ✔
•Equilibrium Dialysis ✔
•Size Exclusion Chromatography (Hummel and Dreyer procedure) ✔
•Analytical Ultracentrifugation
Surface Plasmon Resonance (SPR) ✔
Thursday, 28 October 2010
Point 1... If you’re going to treat the
data with a simple model, make sure
it’s correct
If we are considering drug design against a
protein target, then hopefully the molecule will
bind with high affinity to a single site on the
protein ! Therefore this simple 1:1 binding
scheme will apply
Regardless of experimental technique - we will generally end up fitting the
experimental data with some simple parametric equations yielding estimates for KD
However it’s important to carefully examine the validity of the underpinning binding
scheme or the results will be meaningless ...
Thursday, 28 October 2010
What more complex behaviors might
be encountered?
There could be more than a
single binding site.
The additional sites might
not be equivalent
Binding could be linked to some other
process - like self-association of the
Acceptor.
Thursday, 28 October 2010
Additionally these
sites might not be
independent
(leading to
cooperative
binding).
false plateau and the titration can be ended prematurely.
Point 2: Perform binding studies with an
appropriate concentration of the reactants
The choice of an appropriate value of AT can be illustrated by inspectio
Fig. 6. This figure shows fA as a function of the BT/AT ratio for a system
Kd ¼ 1 mM and diVerent values of AT. In the ideal case (Curve a, AT ¼ 2 # Kd
Kd and the De(AB) values will be well defined and the shape of the binding c
[A]T = 25 x KD = 25 μM
6A@6B@
KD =
= 1 nM
6A.B@
Fraction of the Acceptor bound
1.0
b
[A]T = 2 x KD = 2.0 μM
0.8
a
0.6
fA
[A]T = 0.2 x KD = 0.2 μM
0.4
c
0.2
0.0
Martin and Schilstra. Circular dichroism and its application to the study of
biomolecules. Methods Cell Biol (2008) vol. 84 pp. 263-93
0
2
4
6
8
10
T/AT of Ligand to
MolarBratio
Acceptor
Fig. 6 Titration saturation curves. Observed fractional saturation for a titration of A with B in
Thursday, 28 October 2010
a simple 1:1 complex (AB) is formed (see text). The fractional saturation of A (defined as fA ¼ [AB
The critical points ...
Regardless of the technique used to probe binding, the dissociation
constant KD determines the appropriate concentration range for best
observation of the reversible equilibrium. Generally we want concentrations
of the Acceptor, A, to be a little greater than the KD.
Titrations performed with concentrations of the Acceptor >> KD will make
the stoichiometry of the reaction obvious and are a good check on the
validity of the 1:1 binding scheme.
The binding affinity may partially dictate the choice of technique. e.g.
Interactions with a nanomolar KD are difficult to completely characterize by
ITC, but often well suited for characterization by SPR.
Thursday, 28 October 2010
Studying binding using spectroscopic
techniques
UV absorbance spectroscopy:
Varian Cary 4000
Contact person is Esther Bulloch ([email protected])
CD and fluorescence spectroscopy:
Applied Photophysics Pistar
Contact person is Richard Kingston ([email protected])
Both instruments are located on the 4th Floor of the Thomas Building (Room 427A)
Thursday, 28 October 2010
Studying binding using
spectroscopic techniques
We monitor the interaction of the sample with light in the
UV / visible region.
Absorption spectroscopy: Changes in Intensity
Fluorescence spectroscopy: Changes in Wavelength.
Circular Dichroism spectroscopy: Changes in Polarization
The basic requirement for direct monitoring of binding is
that the spectral signal of the ligand or the protein is
perturbed by binding.
Thursday, 28 October 2010
UV Absorption spectroscopy
Considering the protein:
•
Within a protein the only significant chromophores in the near UV are the aromatic
Tryptophan and Tyrosine residues.
From Biophysical Chemistry. Cantor and Schimmel
•For UV absorption to have utility for binding studies there must be Trp or Tyr proximal
to the ligand-binding site. Unfortunately their absorbance is not particularly sensitive to
structural environment.
Considering the ligand:
•Ligand needs to have some sort of conjugated system present -> excitation energies in
the UV / visible region.
Thursday, 28 October 2010
Fluorescence spectroscopy
Considering the protein:
Tryptophan is the most fluorescent of the protein side chains. Light absorption at
280nm results in light emission around 350 nm
Relative
Fluorescence
•
Trp fluorescence resulting
from excitation at 278 nm
From Protein Structure. A Practical
Approach. Ed T.E. Creighton.
Emission wavelength
•Trp fluorescence is highly sensitive to structural environment, making this an excellent
probe of ligand binding when Trp is located in the binding site.
Considering the ligand:
•As per absorbance ... Ligand needs to have some sort of conjugated system present
Thursday, 28 October 2010
CD spectroscopy
CD spectroscopy measures changes in the polarization of light as it passes through the
specimen.
Considering the protein:
•
The CD signal in the far UV is highly sensitive to the local conformation of the protein
backbone - i.e. to protein secondary structure.
From Protein-Ligand Interactions: Structure and Spectroscopy. Eds Harding & Chowdry.
•For
CD to have utility for binding studies there must be a conformational change
associated with binding.
Considering the ligand:
•Ligand needs to be both chiral and flexible for CD to be of utility.
Thursday, 28 October 2010
The bad news about spectroscopic
investigation of ligand binding ...
• Application
of these techniques will be highly case
dependent. Ignoring the properties of the ligand, they
either require Trp/Tyr near the active site (Absorbance
and Fluorescence) or some significant structural change
associated with binding (CD).
• As
implemented at Auckland we have no automatic
titration system for either the Cary 4000 or the Pistar.
Manual pipetting and sample insertion = low throughput.
Thursday, 28 October 2010
... continued
• There
are no polished software packages for analyzing
spectroscopic binding data. You’ll need to devise your
own solution for fitting the appropriate model to the
experimental data. And be prepared to do a bit of
reading.
• For CD and absorbance spectroscopy the KD will usually
need to be in the micromolar range.
★
Weaker binding necessitates impractically high protein concentrations (the
solution becomes opaque to UV light).
★
Tighter binding necessitates impractically low protein concentrations (Absorbance
or CD signal can’t be reliably measured).
Thursday, 28 October 2010
The slightly better news ...
•
The sensitivity of fluorescence spectroscopy makes it possible to study interactions
with μM - nM KD’s using small amounts of material.
•
The method of choice when it’s possible ???
KD for Adenosine binding was determined
to be 15 μM (Concentration of protein in
the spectrometer cell was 5 μM)
Nucleotide binding to
“PAE2307”
Thursday, 28 October 2010
LOTT ET AL. THE STRUCTURE OF AN ANCIENT CONSERVED DOMAIN ESTABLISHES A STRUCTURAL
BASIS FOR STABLE HISTIDINE PHOSPHORYLATION AND IDENTIFIES A NEW FAMILY OF ADENOSINESPECIFIC KINASES. J BIOL CHEM (2006) VOL. 281 (31) PP. 22131-41
... continued.
• CD
Spectroscopy can give useful clues concerning
structural changes accompanying binding.
CD signal
2782 Biochemistry, Vol. 40, No. 9, 2001
HENKELS ET AL. LINKED
FOLDING AND ANION
BINDING OF THE
BACILLUS SUBTILIS
RIBONUCLEASE P
PROTEIN. BIOCHEMISTRY
(2001) VOL. 40 (9) PP.
2777-89
Thursday, 28 October 2010
on August 3, 2009
i: 10.1021/bi002078y
An extreme example ... the B. subtilis
RNAase P protein binds anions, which
induce the protein to fold
FIGURE 5: Anion-induced folding transitions of the P protein
followed by the change in the far-UV CD signal at 222 nm at 37
°C. Protein samples (5 µM) were initially equilibrated in a 10 mM
sodium cacodylate buffer (pH 7). Anions used in the titration include
tetrapolyphosphate (!), tripolyphosphate ("), pyrophosphate (.),
CTP (3), dCTP (2), CMP (red ~), dCMP (red b), phosphate (red
[), sulfate (red 9), acetate (green 4), fluoride (green ×), chloride
(green 1), perchlorate (green [), and formate (green 0). The total
anion concentration is shown on the x-axis. The lines represent
nonlinear least-squares fits of the intermediate affinity anion titration
data to eqs 8-12, and the parameters are listed in Table 3.
Anion-induced folding
transitions followed by
monitoring the far-UV CD
signal.
First, anions can induce folding through a general ionic
FIGURE 6
protein de
at 222 nm
The P pro
change in
baseline s
concentra
the P pro
to P prote
strength
Furtherm
and wea
series. T
onto ani
Circumventing our basic requirement ...
Thermal and Chemical Shift
experiments.
• In
direct spectroscopic approaches, the spectral
properties of the Ligand or Acceptor must be perturbed
by binding.
• If
this is not the case, then it may still be possible to
study binding spectroscopically using a thermal or
chemical shift assay, where the influence of ligand
binding on protein unfolding is monitored.
Thursday, 28 October 2010
+
Spectroscopic signal
Thermal and Chemical Shift
experiments.
- Ligand
+ Ligand
Temperature or [Chemical Denaturant]
In theory it can be made quantitative but there are a lot of issues - e.g. analysis requires
that the unfolding process is full reversible
Probably best used as a screening technique - using a plate reader it can be made high
throughput.
Thursday, 28 October 2010
Studying binding using isothermal
titration calorimetry (ITC)
MicroCal VP-ITC
Contact person is Richard Kingston
([email protected])
Located in Room 464, Thomas Building.
The ITC measures the heat evolved as two solutions
are titrated.
Thursday, 28 October 2010
Studying binding using isothermal
titration calorimetry (ITC)
One component is loaded into the sample cell, the
other is injected stepwise via an automatically operated
syringe.
Thursday, 28 October 2010
Some Actual Experimental data for a
protein-protein interaction
Raw data
Integrated data and
fitted model
The raw data is integrated, yielding the heat associated with each injection.
The data is usually corrected for the heats of dilution (we’ll come to this in a
minute) before model fitting.
Thursday, 28 October 2010
What do we get from analysis of ITC
data ??
The Equilibrium
constant
governing the
reaction
The breakdown of
the binding energy
into its entropic
and enthalpic
components
€
[ A][ B]
KD =
[ AB]
[ AB]
1
KA =
=
K D [ A][ B]
ΔG =€−RT ln K A = ΔH − TΔS
ITC allows for the complete thermodynamic characterization of binding
€
Thursday, 28 October 2010
Will ITC provide useful information ?
Probably … but low and high affinity binding cause
problems with model fitting ...
Too weak
Just Right
Too Strong
N.B. Useful
range of
analysis can be
extended at the
high affinity
end, if a weak
competitive
inhibitor of
binding is
available
(displacement
analysis)
KD = 10-4 M
Can’t determine enthalpy
reliably
Thursday, 28 October 2010
KD = 10-6 M
KD= 10-8 M
Can’t determine affinity
reliably
Heats of dilution ... critical corrections
to the raw data
Binding data obtained by ITC have to be corrected for the heats which result simply
from dilution of the reactants (i.e. that have nothing to do with the binding process).
These heats can be readily determined experimentally, by injecting the syringe
component into buffer, and buffer into the cell component.
Typical binding isotherm (top)
and heat of dilution associated
with the syringe component
(bottom)
Notice the convergence of the
titration and the control, once
binding is saturated.
From Tame, O’Brien & Ladbury (1998) Isothermal Titration Calorimetry of BioMolecules. in BioCaloimetry: Applications of Calorimetry in the Biological Sciences. Chowdhry
ed.
Thursday, 28 October 2010
ITC ... The Pros
•The closest thing we have to a universal technique for
studying binding - almost all binding reactions consume
or generate heat.
•ITC is the only technique which allows direct estimation
of the binding enthalpy. This facilitates the full
thermodynamic characterization of binding.
•Microcal’s data analysis software is adequate for the
task, allowing a non-expert to pretty quickly get reliable
numbers.
Thursday, 28 October 2010
ITC ... The Cons
•Extremely demanding on material. The calorimeter cell
has a volume of 1.4 mls !!! (However ITC is nondestructive, so you can recover the material).
•Quite time-consuming and fiddly to perform
ITC ...Overall
•Probably the gold-standard when it comes to binding
studies, but a poor choice for high throughput
applications, or highly exploratory initial studies.
Thursday, 28 October 2010