Лекция 6 Методы исследования взаимодействий с участием белков (Co-IP,

ITC, MST, SPR, BLI, QСM). Примеры.
Случанко Н.Н.

proteins (PMID: 17640003)
For each protein ~10 protein partners (interactome)
Human “interactome” -

biochemistry would appear to be largely run by a set

interactions: • Different proteins • Enzyme – inhibitors • Antibody – antigen • Protein

calorimetry (ITC) • Surface plasmon resonanse (SPR) • Quartz microbalance (QMB) • Fluorescence

https://link.springer.com/book/10.1007%2F978-1-4939-2425-7

and detection of bound partner proteins
Reverse
Immunoprecipitation of partner proteins

Ge et al, J.Proteom.Res., 2010: 5848-5858

is free ligand
ML is complex
Lo >>

Case 1 (specific)

Case 2 (general)

https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindderveq.html

Lo >> Mo, you can’t measure Lfree

ML =

Mo * Lfree

KD + Lfree

hyperbola

parabola

KD =

(Mo – ML) * (Lo – ML)

ML

(just put your numbers for Ao, Bo, KD and choose

(just put your numbers for Ao, Bo, KD and choose

Root 1

Root 2

= [M]2/[D]
[Mo] = [M] + 2[D]
 [M] = [Mo] -2[D]
Kd =

Lo >> Mo => quadratic equation

= 1/Keq
ΔGo = - R T ln Keq = R

25 µM

= 25*10-6 M

ΔGo = R*T * (-10.6) =

@ 20 °C

2 cal/mol*K

– 6.2 kcal/mol

= 1/Keq
ΔGo = - R T ln Keq = R

25 nM

= 25*10-9 M

ΔGo = R*T * (-17.5) =

@ 20 °C

2 cal/mol*K

– 10.3 kcal/mol

= 1/Keq
kon
koff
Von = Voff
kon [A] [B] = koff [AB]

koff / kon = [A] [B] / [AB] =

(FP)
Isothermal titration calorimetry (ITC)
Microscale thermophoresis (MST)
Surface plasmon resonance (SPR)
Biolayer interferometry

membrane separates the two chambers
MW cutoff of the membrane allows

M

Chamber 1

Chamber 2

is calculated
M total is
known

ML
Fast
Easy
Inexpensive
Accurate determination of affinity (KD) and stoichiometry of interaction
Membrane type

Features

(AChE)
AChE

size of the particle bearing a fluorophore

concentration
P has physically possible values ranging from –0.33 to 0.5

=

2 P

3 – P

temperature, R = gas constant and V = molecular volume

rotational correlation time of the dye:

Simulation of the relationship between molecular weight (MW) and fluorescence polarization (P)

Φ is found to increase by ~1 ns per 2400 Da increase of MW

dyes with various fluorescence lifetimes (τ)

Perrin equation (1926):

Fundamental P (Po) ~0.5 (max)

detection
Rapid response
Real-time (not only equilibrium studies)
Kinetic analysis (association/dissociation) is possible
Separation

becomes warmer than the reference cell
• Binding causes a downward

calculated

stoichiometry, association constant, and binding enthalpy) in a single experiment
Modification

Disadvantages:
Large sample quantity needed
Kinetics (i.e. association and dissociation rate constants) cannot be determined
Limited range for consistently measured binding affinities
Non-covalent complexes may exhibit rather small binding enthalpies since signal is proportional to the binding enthalpy
Slow with a low throughput (0.25 – 2 h/assay), not suitable for HTS

sample (method is entirely optical and contact-free)
Ability to measure complex

Disadvantages:
Hydrophobic fluorescent labelling required, may cause non-specific binding
No kinetic information (i.e. association and dissociation rates)
Highly sensitive to any change in molecular properties

times above KD)
Accurate protein concentration must be determined
Zero concentration should

https://www.youtube.com/watch?v=e_tNkxbE2kY

binding model
Biacore

kinetics and thermodynamics)
Medium throughput
Sensitivity and accuracy
Measures over a very wide

Disadvantages:
Expensive instrument and sensors
Expensive maintenance
Steep learning curve
Specialized technician or senior researcher required to run experiments
Immobilization of one of the binding partners required

sample compatibility
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106882
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089413/
Exemplary studies:

compatibility
Fluidic-free system so less maintenance needed
Disadvantages:
Immobilization of ligand to surface of

(5-10 MHz) with the Au chip
Mass detection with super accuracy

https://openqcm.com/openqcm

Sauerbrey equation:

https://www.youtube.com/watch?v=xDKOUpSR3EQ

Xdelic

measured
https://www.youtube.com/watch?v=xDKOUpSR3EQ

by their sequence
Spectroscopy methods
Stability of proteins
Protein structure – high resolution