1. Methods: Protein-Protein Interactions
Biochemistry 4000
Dr. Ute Kothe

2. Remember: PC of bindingk1
k-1
P + L P-L
Equilibrium dissociation constant
KD: concentration of 50% binding
[P] [L] k-1
KD = =
[PL] k1
Fraction bound (F):
[PL] [L]
F = =
[Ptotal] [L] + KD

3. Electrophoretic mobility shift assay
Also band shift
or gel retention/retardation assay
Detection of
nucleic acid – protein interaction
protein – protein interactions
Pre- Incubation of biomolecules to form complex
Native PAGE allowing the interactions to be maintained during electrophoresis
Coomassie Autoradiography
DNA
Full-length protein
truncated protein

4. Electrophoretic mobility shift assay
Analysis:
Coomassie stain
Ethidium bromide stain of nucleic acid
Autoradiography to detect radioactivley labelled proteins or nucleic acids
Shift of bands relative to free components indicates interaction
Advantages:
qualitatively detection of interaction
low cost: no special equipment needed, low amounts of biomolecules
Disadvantages:
No quantitative data (in the best rough estimation of KD)
Interacting biomolecules must have different electrophoretic mobilities

5. Generates monochromatic beam
Light Scattering
Similar to Scattering of X-rays from a crystal
But: UV or VIS light, i.e. wavelength is larger than size of biomolecule
No determination of structure, but of size (molecular weight)
Size reflects formation of oligomers
or complex with other protein
Instrument:
Compare intensity of incident light with light scattered at a given angle θ
Solutions must be well filtered to avoid scattering from large dust particles!
Generates monochromatic beam

6. Light Scattering - Principle
Simple example: monochromatic, linearly polarized light interacting with a single molecule
The electric field of the light oscillates at the point of the single molecule
causes the molecule to have an oscillating dipole
oscillating dipole acts as mini-antenna dispersing some energy in
directions other than the direction of the incident radiation
= elastic Rayleigh SCATTERING

7. Static Light Scattering
Scattering is dependent on
l, θ, and concentration – can be chosen
refractive index – measure for
polarizability in visible region of
light, can be measured
the molecular weight MW.
Determination of molecular Weight MW of a monomner (A) by measuring at various concentrations and extrapolating to c=0 (to account for solution nonideality).
What about Dimers (B)?
How?
Depending on the measuring angle θ, the original intensity I0 and the measured scatter intensity i0 the parameter Rθ is calculated
constant K is dependent on wavelength l, refractive index and the specific refractive index dn/dC (can be measured)

8. Dynamic Light Scattering
Instead of measuring the average light scattering in a large volume, a small volume is observed.
Fluctuations in local concentrations
over time become significant
reflect diffusion of molecules
can be used to determine
diffusion coefficient D

9. Surface Plasmon Resonance (SPR)
Sensor chip with gold film:
carries Protein 1
Protein 2 (interaction partner) is introduced in flow channel (constant flow)
Binding interaction changes mass at surface of chip
Refractive index of chip changes
reflection angle and intensity of polarized light changes

10. SPR: Principle Principle:
Total reflection occurs at the critical angle which depends on the refractive index of the surface.
Energy carried by photons can be transferred to electrons in a metal at a certain wavelength (resonance)
At the resonance wavelength, almost all light is absorbed.
This creates a plasmon, a group of excited electrons in the metal surface which behave like a single electrical entity.
The plasmon generates an electrical field about 100 nm above and below the surface, called evanescent wave.
Characteristic used to measure binding:
Change in chemical composition of environment of plasmon field causes a change in refractive index and thus in the resonance wavelength / in the critical angle for total reflection.
Change in mass of complex bound on surface is proportional to change in angle of totally reflected polarized light.

11. SPR: Results Typical Sensogram
dissociation constant (KD) from signal intensity in dependence of ligand concentration
apparent association and dissociation constants (kon, koff) from signal change during injection of ligand / removal of ligand
Disadvantage:
No equilibrium method
Constant flow of ligand

12. Isothermal Titration Calorimetry (ITC)
Determine absorption or release of heat (q) upon binding of a
ligand to a biomolecule
heat is proportional to enthalpy DH°(T) and number of moles
complex (nPL = V * [PL]): q = DH°(T) * V * [PL]
[L]
q = DH°(T) * V * [Ptotal]
[L] + KD
By measuring q at various ligand concentrations while knowing
the volume and total protein concentration,
DH°(T) and KD can be determined!
Remember: DG°(T) = - RT ln KA and DG°(T) = DH°(T) – T*DS°(T)
DG°(T) and DS°(T) can be determined!

13. ITC - Instrument stepwise addition of ligand into protein
solution of known concentration
by comparison with a reference cell
containing only buffer, the energy is
measured which is required to maintain
a constant termperature over time
heat q is obtained by integrating peak
area over time
Disadvantage:
Significant amounts of proteins needed
(Size of cell 1 – 2 ml)
Tight binding interactions can not be studied (KD should be in µM range)

14. ITC - Data
Binding between core binding domain of exterior glyco-protein gp120 form the HIV-1 virus and the CD4 receptor fo the target host cell
DH° = -263 kJ/mol,
KA = 5 x 106 M-1

15. Other Methods Fluorescence (FRET) Size Exclusion Chromatography
Immuno precipitation
Affinity chromatography
Crosslinking
Analytic ultracentrifugation
Mass spectrometry