Methods:
Protein-Protein Interactions
Biochemistry 4000
Dr. Ute Kothe
Remember: PC of binding
k1
P+L
P-L
k-1
Equilibrium dissociation constant
KD: concentration of 50% binding
[P] [L]
k-1
KD =
=
[PL]
k1
Fraction bound (F):
[PL]
[L]
F=
=
[Ptotal]
[L] + KD
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
DNA
Full-length protein
Autoradiography
truncated protein
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
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
Light Scattering - Principle
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
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)?
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
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
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.
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
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]
q = DH°(T) * V * [Ptotal]
[L]
[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!
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)
ITC - Data
Binding between core binding
domain of exterior glycoprotein 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
Other Methods
• Fluorescence (FRET)
• Size Exclusion Chromatography
• Immuno precipitation
• Affinity chromatography
• Crosslinking
• Analytic ultracentrifugation
• Mass spectrometry