Protein-Ligand Interaction 2

Question 1

Dissociation Constants of Different Techniques

Answer 1

• NMR and SPR have the lowest ranges down to 10mM
• DSF down to 1mM
• ITC down to 100 microliters

Question 2

Isothermal Titration Calorimetry

Answer 2

• thermodynamic measure of protein binding
• enthalpy change on binding results in release or absorption of heat
• measure heat changes as a ligand is titrated into a protein sample in a calorimeter
• changes observed as titration proceeds are related to binding affinity and stoichiometry of reaction

Question 3

ITC Instrument

Answer 3

• sample cell is put in a shield to prevent external heat changes
• ligand is titrated it and tip mixes ligands as it is titrated

Question 4

ITC Data

Answer 4

• Measurements consist of the time-dependent input of power required to maintain equal temperatures between the sample and reference cells.[citation needed]

In an exothermic reaction, the temperature in the sample cell increases upon addition of ligand. This causes the feedback power to the sample cell to be decreased (remember: a reference power is applied to the reference cell) in order to maintain an equal temperature between the two cells. In an endothermic reaction, the opposite occurs; the feedback circuit increases the power in order to maintain a constant temperature (isothermic/isothermal operation).[citation needed]

Observations are plotted as the power needed to maintain the reference and the sample cell at an identical temperature against time. As a result, the experimental raw data consists of a series of spikes of heat flow (power), with every spike corresponding to one ligand injection. These heat flow spikes/pulses are integrated with respect to time, giving the total heat exchanged per injection. The pattern of these heat effects as a function of the molar ratio [ligand]/[macromolecule] can then be analysed to give the thermodynamic parameters of the interaction under study.

Question 5

ITC Fitting Data

Answer 5

• by measuring the heat change per mole of injectant and knowing the V0, ligand and protein concentrations, we can fit the data to an equation and determine Kd

Question 6

ITC Experimental Considerations

Answer 6

• ligand concentrations should be higher than the protein concentrations
• need to dialysise and degass components to avoid dilution
• need to know concentrations accurately
• product of protein concentration and binding constant must be lower than 1000 for accurate measurement

Question 7

ITC Parameters

Answer 7

• only technique yielding binding constants, reaction stoichiometry, binding enthalpy to provide a complete thermodynamic profile
• can determine G/S from this data and determine the type of energetic contribution driving a binding event
• even if reactions have the same overall energy change (G) the different enthalpy and entropy components have different effects

Question 8

Evaluation of ITC

Answer 8

• accurate measurement
• determines binding constants and stoichiometry
• no reagent immobilisation
• no chemical modification
• need large protein amounts
• high throughput

Question 9

Surface Plasmon Resonance

Answer 9

• used in binding assays/drug design
• directly observes binding kinetics
• measures Kon and Koff

Question 10

Principle of SPR

Answer 10

• light incident on a thin metal film induces a surface plasmon : wave of electron density in the film
• at a critical reflection angle the reflected light has minimum intensity (photon absorption is maximal)
• value of resonance angle depends on refractive index of the substrate material

Question 11

SPR Setup

Answer 11

• bait molecule is attached to the thin gold film via a tether (usually biotinylated protein)
• solution of prey molecules flows past
• on binding the refractive index of the interface increase, displacing the angle of reflected intensity minimum to give a measure of binding
• 1ng of bound protein per mm of surface are changes the resonance angle by 0.1 degrees
• allows direct observation of association and dissociation (and Kd from curve slope)
• association increases resonance units and vice versa

Question 12

Evaluation of SPR

Answer 12

• quantitative
• accurate
• sensitive
• direct kinetic visualisation
• expensive
• need to optimise conditions
• one binding partner is not in the solution phase : realistic??

Question 13

Chemical Shift Perturbation

Answer 13

• CSPs are related to changes in chemical environment of amide or CH groups
• ligand binding can induce changes in chemical shifts
• best technique for measuring weak interactions
• must control pH to prevent amide proton exchange

Question 14

CSP analysis

Answer 14

• measure reference spectra of protein and add ligand then measure again
• backbone resonance assignments of protein allow comparasion
• observe step wise movement to a saturated position
• use CSP to identify interaction surfaces and binding information without needing to know protein structure
• cannot separate CSP from direct binding or from allosteric changes
• can correlate the N and CH dimensions to identify values considered to be in the binding site
• ** see notes ***
• can obtain residue specific dissociation constants and Kon/off

Question 15

NMR Timescales

Answer 15

• resonance of an atoms changes when interacting with ligand
• visible in 2D spectra
• weak vs. strong binding creates different patterns
• can compare directly Koff and chemical shift
• predict the interaction type by the spectra appearance

Question 16

Proton/Deuterium Exchange

Answer 16

• measure reference spectrum then add ligand
• lyophilise to remove water
• dissolve in deuterium
• observe decrease of signals due to amide exchange
• amide protons in the binding interface are protected so do not exchange (compared to reference)

Question 17

Cross Saturation

Answer 17

• protons of aliphatic protons in unlabelled protein 2 are saturated with NMR pulse
• via cross relaxation the magnetization moves to labelled protons of protein 1
• signal intensity of selected amides in protein 1 decreases if in contact with the protein 2 surface
• define the interaction surface via amide signals with reduced intensities
• so the magnetization only transfer via direct contact with protons of protein 1 (i think***)

Question 18

Identification of Interaction Surfaces

Answer 18

• effect of saturation time on the intensity ratios of cross-peaks originating from the backbone NH groups with irradiation to those without irradiation
• if an amide is in the binding site the resonance is saturated and the intensity decreases over saturation time
• high level of deuteration in solvent improves intensity ratio by decreasing spin relaxation

Question 19

Cross saturation exam answer

Answer 19

Protein A, a fully deuterated, 15N-labeled protein (whose amide deuterons have been exchanged with solvent protons), is incubated with unlabeled protein B. The aliphatic protons (0-3ppm) of protein B are selectively irradiated, leading to cross saturation of nearby protons (including aromatics and amide protons, 6- 10ppm). Protein A is not directly affected by this irradiation, but magnetization can be transferred from B to amide protons of A at the AB interface, to become observable in an 15N HSQC spectrum. As in the previous cases, backbone resonances can be used to map a ligand binding site in the labelled protein. This technique works best in systems with ligands strongly bound to target proteins.

Question 20

Timescales

Answer 20

Describe fast, intermediate and slow regimes of exchange in NMR timescales. In fast exchange rates at NMR timescales, dissociation constants (KD) can be measured, assuming that ligand binding is diffusion controlled (kon ≈ 109 M-1 s-1). In this exchange regime, measurable KD‘s are > 5-10μM. In fast exchange, the NMR signals involved in protein-ligand interactions are observable during the titration as the weighted average of the free and ligand bound forms. These chemical shift perturbations can be used to obtain the kD of the interaction using an equation with a quadratic form and non-linear least squares fitting. In the intermediate or slow exchange regimes, line broadening indicates ligand binding, but KD‘s are not measurable.
- These chemical shift perturbations can be used to obtain the kD of the interaction using an equation with a quadratic form and non-linear least squares fitting.