Quantitative analysis of protein-protein interactions lablifeb.org Ctirad Hofr LifeB - Laboratory of interaction and function of essential Biomolecules Functional Genomics and Proteomics National Centre of Biomolecular Research II U l\l I SCI IOverview of quantitative methods for analysis of protein-protein interactions Theory = basis for practice Binding curve, equilibrium dissociation constant, linear range of the detector. Fluorescence leads in numbers, but you can do without it Determination of binding affinity of fluorescently labeled proteins - fluorescence anisotropy, microscale thermophoresis, detection of binding of molecules immobilized on the surface - surface plasmon resonance; study of binding of unmodified proteins directly in solution - isothermal titration calorimetry. Which is best - comparison of methods Summary of the practical advantages and disadvantages of quantitative methods for protein-protein interaction analysis. lablifeb .org f I U 111 I SCI Binding curve 1 isotherm To bind two proteins A,B and form an A.B complex at constant temperature A+ B A.B we define equilibrium constants association and dissociation Ka = [AB] [A][B] KD = [ARB] [A'B] If protein B is added sequentially to protein A, the binding curve can be expressed by the equation Y = % bound = [B] [B]+KD 100% lablifeb .org Dependence of the binding rate on the total concentration of added protein B 100 8 75 o o E 50 TD o -Q >o 25 0 K 20 40 60 Protein concentration [B] 100 KD dissociation constant - the protein concentration at which exactly half of the molecules are bound fI U II I SCI Binding curve 2 origin of the equation The binding rate is expressed as the ratio of the concentration of complex A.B to the total concentration of protein A multiplied by 100%. Dependence of the binding rate on the total concentration of added protein B 100 ^ 75 Y = % bound = [A-B] A 100% TOT =3 O _(D O E "D C 13 O .Q •vP K A = K After substituting A.B to AT0T we get the equation for the binding curve [A - B] = KA[A][B] AT0T = [A'B] + [A] 50 25 0 0 20 40 60 80 Protein B concentration [B] % bound = [B] [B] + KD 100% 100 lablifeb The binding curve is a hyperbola with the value .org of the dissociation constant KD in the denominator. M U III SCI I Binding curve 3 - | logarithm of concentration = sigmoid If protein A is added to protein B over a sufficiently wide concentration range, the binding rate versus logarithm of the concentration of B is a sigmoid. % bound = [B] [B] + KD 100% Dependence of binding rate on logarithm of the total concentration of added protein B 100 75 o 50 _Q KD dissociation constant - inflection point of sigmoid Slope of the sigmoid - a measure binding cooperativity when multiple proteins B bind to a single protein A. lablifeb .org How does binding jfC^ cooperativity change the shape of the sigmoid? 25 0 J / -1 ■C https://en.wikipedia.org/wiki/l-l ilLequation (biochemistry) 0.1 1 10 100 1000 Logarithm of protein concentration log 10 [B] fI U II I SCI Know your detector - linear detector range = accurate quantitative measurements Overall, the detection curve has a sigmoid shape. For accurate quantitative measurements, it is essential that the increment in signal is directly proportional to the increment in concentration of the protein-protein ° complexes. 8 CD This is fulfilled for the linear range of the detector I- the detection region, where an increase in concentration, for £ example, doubles the signal value. o Area below the linear range - we are close to the minimum detection limit = non-linear response. Area above the linear range - the detector is overwhelmed with signal - saturated, a large change in concentration will cause a relatively small and non-linear increase in signal. CO 00 Saturation of detecto Concentration Always find out the linear range of the detector. For quantitative measurements it is essential that the measurement values are within the linear range of the detector. Fluorescence anisotropy-based determination of binding affinity Fluorescence anisotropy - principle The "directionality" of the emitted light increases after the formation of a protein-protein complex; after excitation by linearly polarized light, the fluorescence of the labelled protein-protein complex is emitted predominantly in one direction. Practically - we label the smaller of the proteins. - It is sufficient to label 100 mg of protein in a cuvette. - added larger protein is not labelled, -total concentration of the added protein is at least 10 times greater than the concentration of the labeled protein. Excitation in one direction A Time from excitation tor emission of fluorescence Fluorescence emission low anizotropy t High anisotropy ..directionality" 0 0 100 75 TD C o 50 _Q a*-r\ r- i_ Ö 8 25 0 i Composition of solution in cuvette Concentration of a 100 Fluorescence anisotropy measurement setup r = hi-1!. Wv-Wh //7 + 2/j_ IVV+2IVH analyzer detector The value of anisotropy r is the ratio of the difference of lvv - lVH fluorescence intensity at parallel (vertical) and perpendicular (horizontal) rotation of the emission analyzer to the excitation polarizer and the total fluorescence intensity Ivv+2'vh in 3D - all three directions of fluorescence propagation. Ilablifeb .org polarizer Practically - Instrument - fluorometer with polarizer of excitation light and rotatable polarizer = analyzer of emitted fluorescence to detect intensity in different directions. - Required ~1 Ox higher concentration than normally used for measuring conventional fluorescence - polarizers transmit lux less light. - Anisotropy r is a dimensionless without unit (ratio of numbers). mi u i\i i SCI IMicroscale thermophoresis MST Principle of MST We generate a local temperature gradient - irradiate the sample in the capillary with an infrared (IR) laser. At the same time, we illuminate the sample with excitation light for the fluorophore that labels the smaller protein. We detect the movement of the fluorescently labelled molecules as a change in fluorescence in the micro-region illuminated by the IR laser. At a constant concentration of the fluorescently labelled protein, we increase the concentration of the added unlabeled protein = ligand. We observe a decrease in the rate of fluorescence decrease over time with increasing ligand concentration. I lablifeb https://en.wikipedia.org/wiki/Microscale_thermophoresis Sample irradiation time ~ 20 s H/| U 111 I SCI o B 900 700 IR-laser on Practical MST measurement of protein-protein interactions Label fluorescently a smaller protein-analyte. Create a dilution series of the second protein = ligand by diluting it 2 times. Mix the solutions so that the concentration iaoo of the labeled protein is the same, but the concentration of the ligand varies by 5 orders of magnitude. Aspirate the samples into capillaries (5uL). Measure the change in fluorescence after switching on the IR laser. Plot the change in fluorescence versus the logarithm of the ligand concentration. From the inflection point of the sigmoid, determine the dissociation constant of the protein-ligand complex formation. lablifeb .org Ligand -is m Fluorescent molecule Saturation f IR-laser off 201 %o) 15- E o 10- c LL 5- Baseline 0- I I I I I I io'4 10: i i i inii|—i i 11ini|—i 111 11|—i 1111iu|— ' 0.01 0.1 1 10 Ligand (nM) Kindly provided by Dr. Josef Houser, CORE FACILITY Biomolecular Interactions and Crystallization http://bic.ceitec.cz/cs n/i u in i SCI MST - Microscale Thermophoresis Glass capillary IR laser OFF A 1 1. 2. Initial state - initial fluorescence of sample. Thermophoresis - change in fluorescence due to the thermophoretic motion of molecules. 3. Steady state - local concentration of molecules decreases in the heated region until it reaches a steady-state distribution. Back-diffusion - fluorescence recovery driven by mass diffusion of molecules. 4. Time Prepared by Tomáš Janovic Analyzing protein-protein interaction with MST Measuring MST for ligand serial dilution Evaluating interaction affinity * F Time o D Ligand concentration log [L] Prepared by Tomáš Janovic Protein-protein interaction on Surface plasmon resonance - Principle At the transition between the glass and the gold layer, ideally 50 nm thick, the light - laser is reflected. At the resonance angle 0, an increased absorption occurs, which is detected by the detector. An evanescent (disappearing) wave of resonant electrons = plasmons is produced, which gradually decays with distance from the surface. The range of the evanescent wave is approximately 100 nm into the solution space. Plasmons are very sensitive to changes in the environment in which they move. Thus, SPR allows the detection of changes due to the binding of proteins on the surface of the gold layer. The gold is coated with dextran, on which one interacting protein is immobilized. A second protein is added to a buffer that washes the surface with the immobilized protein. Once the protein-protein complex is formed on the surface, a change in the resonance angle 6 is detected. We observe the kinetics of complex formation in real time. the surface SPR Evanescent wave of plasmons - resonating electrons on the surface = surface resonance plasmons that absorb part of the light O 7? o o . 0 , o Light source laser I i_ Au 50 nm T The resonance angle at which plasmons are created Prism - glass flow SPR measurement protocol Immobilize protein L - ligand on SPR chip. Saturate the binding sites on the surface without immobilized protein L. Wash the chip with buffer. Wash the chip with the second protein A - analyte. Detect signal change. The initial part of the curve shows the kinetics of the association of the two proteins. The final part of the curve describes the dissociation kinetics of both proteins. By fitting the binding models, we determine the values of the association rate constant ka and dissociation rate constant kd The association rate constant ka describes the rate of complex formation, i.e. the number of LA complexes formed per second in a one molar solution of L and A. The units of ka are M1s1 and are typically between 1.103 and 1.107 in biological systems. The dissociation rate constant kd describes the stability of the complex, i.e. the fraction of complexes that decays per second. The unit of kd is s_1 and is typically between 1.10-1 and 1.10-6 in biological systems. A kd of 1.102s1 = 0.01 s_1. This means that 1 percent of the complexes decay per second. Associoation Saturation -Equlibrium Dissociation .0 o flow 0 o 0 0 o ® C 0 © (Do o o (2) (0) o a;