Methods:Protein-Protein Interactions

Biochemistry 4000

Dr. Ute Kothe

Remember: PC of bindingk1

k-1

P + L P-L

Equilibrium dissociation constantKD: concentration of 50% binding

[P] [L] k-1KD = = [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

DNA Full-length protein truncated protein

Coomassie Autoradiography

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

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!

• 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

Generates monochromatic

beam

Light Scattering - PrinciplePrinciple: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• , 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: PrinciplePrinciple: 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

• 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 methodConstant flow of ligand

Typical Sensogram

Isothermal Titration Calorimetry (ITC)

Determine absorption or release of heat (q) upon binding of a ligand to a biomolecule heat is proportional to enthalpy H°(T) and number of moles complex (nPL = V * [PL]): q = H°(T) * V * [PL]

[L]q = H°(T) * V * [Ptotal]

[L] + KD

By measuring q at various ligand concentrations while knowing the volume and total protein concentration, H°(T) and KD can be determined!

Remember: G°(T) = - RT ln KA and G°(T) = H°(T) – T*S°(T)

G°(T) and S°(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 glyco-protein gp120 form the HIV-1 virus and the CD4 receptor fo the target host cell

H° = -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