Protein-Ligand Binding_L4 Flashcards
list the Kd ranges that the following technique can measure:
(1) protein-observed NMR
(2) ligand-observed NMR
(3) surface plasmon resonance(SPR)
(4) enzyme/binding assays
(5) differential scanning fluorimetry(DSF)
(6) isothermal titration calorimetry(ITC)
(1) protein-observed NMR: 10mM-1nM
widest range of affinity
(2) ligand-observed NMR: 10mM-1μM
(3) surface plasmon resonance(SPR): 5mM-1nM
(4) enzyme/binding assay: 500μM-1nM
(5) differential scannning fluorimetry(DSF):1mM-1nM
range reduced to be more sensitive to protein/ligand aggregation
(6) isothermal titration calorimetry(ITC):500μM-1nM
principles of the isothermal titration calorimetry
(1) A thermodynamic measure of protein binding
(2) Enthalpy change (DH) on binding (at constant T and P) that results in release or absorption of heat (exo- or endothermic)
(3) We can measure heat changes (+ve or -ve) as a ligand is titrated into a protein sample in a calorimeter
(4) The changes observed as the titration proceeds are related to the binding affinity and stoichiometry for the interaction
What is the basic setup of the isothermal titration calorimetry?
(1) ligands are injected into the sample cell which is maintained at the same condition as the control cell, the control cell contains only water and buffer at constant pH and temperature
(2) the instrument is isolated to avoid the heat exchange between the exterior environment
(3) the ligands are introduced into the sample cell by the syringe, propeller connected to the syringe mixes the sample fully to reach the solution equilibrium.
(4) before the experiment is setup, ~10-50mM is the typical starting macromolecule concentration in the reaction cell (normally 0.2 or 1.8ml volume, but the volume depends on the setup provided). Optimisation of the experiment may require several ITC scans, but material is easily recoverable as technique is non-destructive.
(5) Ligand (titrant) stock concentrations should be ~10 to 20 times higher than concentration of [M]
(6) All interacting components must be degassed and dialysed extensively in the same buffer to avoid large heats of dilution/mixing which interfere with the reaction. It is recommended to dissolve the ligand in final dialysis solution
(7) Accurate and reproducible ITC data require careful determination of [M] and [L] concentrations (e.g. by UV/VIS absorbance from known molar extinction coefficients: better if two approaches are used for the two parameters)
How will be ITC raw data be presented?
(1) heat change, Q is plotted on the y-axis against the time on the x-axis
(2) three binding phases are observable on the graph:
1. Initial phase [L] «_space;[P], the amount of the ligands are far less than that of the protein and all the ligands are bound to the protein
2. intermediate phase: the enthalpy change per injection decreases because the binding sites start to be filled in with the ligands.
3. end phase: all the binding sites are saturated with the ligands, no further binding.
The equation calculating the enthalpy change from the heat change for proteins of single binding site
(1) Q=V0ΔHPt
(2) Q: heat change
V0: volume of the sample cell
ΔH: enthalpy change of binding per mole of complex formed(J/mol)
[Pt]: concentration of total protein
(3) the enthaply change can be deduced from the equation where the Q is measureable
(4) ITC graph can be compared with the dilution of ligands where no protein is present, in order to substract the ligand influence from the sample. The heat change-time plot can be translated to the enthaply changed per mol of injection versus the molecular ratio of the injection mole over the protein mole. The gradient at the halfway of the curve is equivalent to the Kb (binding constant)
(5) The product of the protein concentration in the calorimeter cell [P], and the binding constant KA, a parameter known as c, (also called sigmoidicity) must be lower than 1000 for the reaction to be measured accurately by ITC ( c= [P]KA ).
In practical terms, this restriction sets an upper limit of ~109 M–1 for KA(KD = 1nM), the limit of the Ka is also a shortcoming for the instrument affnity range. Moreover, the rapid jump in the heat change results in larger error
What parameters can be obtained from ITC?
(1) Gibbs free energy
(2) enthalpy change/heat change
(3) Ka/Kd/c
(4) entropy change
(5) the stoichiometry(the number of binding sites on the protein)
(6) thermal discrimination can therefore be constructed from the entropy change, enthalpy change and Gibbs free energy to reveal which term contributes to the favourable binding of ligands to proteins
pros and cons of ITC?
(1) pros:
1. Accurate measurement
2. Can determine binding constants and stoichiometry:
DH, DG, DS, n and KD
3. No immobilisation of reagents required
4. No chemical modification of reagents
5. Can be high throughput - with suitable equipment
(2) cons:
1. relatively large quantity of protein input required(0.2-1.8ml at 10-50mM, as starting test concentrations)
2. Not accurate if affinity is too tight (nanomolar KD): the problem can be solved by adding inhibitors in some cases.
3. Relatively expensive apparatus
4. rapid change in the heat change is more error-prone
What parameters can be measured from the surface plasmon resonance(SPR)?
(1) measure kon and koff
What principles is SPR based on?
(1) Light incident on a thin metal film induces a “surface plasmon” - a wave of electron density in the gold film
(2) At a critical reflection angle, the reflected light has minimum intensity (condition for absorption of photons by the plasmons is maximal - resonance)
(3) The value of the resonance angle depends on the refractive index of the substrate material. The more light being refracted, the less light being reflected
What will the experimental setup be like?
On binding to the bait proteins(by the tether) attached at the gold film, the refractive index at the interface increases - this displaces the angle of the reflection intensity minimum, giving a measure of binding, as the plasmon-induced evanescent extends beyond the gold film. The detector detects most of the light at the two sides rather than at the centre, thereby a region of darkness is observed. If the refractive index value increases, the location of the darkness shifts away fro, the centre
data from SPR?
(1) 1000 RU = 1ng of bound protein/mm2 of chip surface area
(2) 1000 RU is equivalent to the change in 0.1° degrees in the resonance angle.
(3) Formation of the protein-ligand complex shifts the resonance angle.
(4) This can be followed in real time.
(5) Allows direct observation of association and dissociation.
(6) The resonance angle remains minimal from time 0 until the prey proteins are added into. The resonance angle increases with the number of bound ligands to the satuarion, the angle decreases again as the buffer removes the bound ligand off, so then angle value falls down to 0 gradually.
(7) Kd=Koff/Kon
Traces show an increase in resonance units (RU) when two binding partners associate and a decrease in resonance units when they dissociate.
The concentration of the binding partner in the mobile phase (analyte) was varied from bottom to top as follows: 0.1 μM, 1 μM, 10 μM, and 100 μM.
Dissociation constant KD = 26.4 μM.
Max height of signal is proportional to total mass bound - info on stoichiometry
pros ans cons of the SPR?
(1) Pros
1. Quantitative
2. Accurate
3. Sensitive: small quantities needed (µg);
4. Direct visualisation of kinetics
(2) Cons
1. Expensive equipment
2. May take some effort to optimise conditions – attachment to chip
3. One binding partner not in solution phase – validity of KD?
introduce nuclear magnetic resonance NMR
(1) Modern methodology – in constant development
(2) Series of techniques to identify PLI’s: combine binding constants and structural information
(3) Able to observe/measure interactions with a wide range of affinities
(4) Also for non-crystallisable complexes or as complement to XRD/EM studies
What is chemical shift perturbation(CSP)?
(1) 15N-1H HSQC: more widely used than 13C-1H HSQC because it is more susceptible to environmental changes, e.g. proton exchange, pH changes
(2) 13C-1H HSQC: ligand binding induces the changes in the chemical environments for the groups.
(3) Chemical shift perturbations (CSPs) are related to changes in chemical environment of amide groups (e.g. 15N-1H HSQC spectra) or CH groups (e.g. 13C-1H HSQC spectra).
(4) ligands can induce chemical shift changes, ligand binding induces the different responses of the chemcial groups to the environment.
(5) CSP analysis gives residue-specific information, peaks migration is observable on the spectrum between the two conditions where no ligand is bound and the ligand is saturated on the protein. The interacting surface can be defined (i.e. CSP data plus backbone resonance NMR assignments - from analysis of 3D spectra). If the protein is assumed to be a rigid body, it will be confident to tell which residues participate in the catalysis. It is not possible to separate CSPs from direct ligand binding and CSPs due to allosteric/ conformational changes upon ligand binding (possible with other NMR techniques).
(6) If the structure or model of the protein is not available, the homologous protein structure can be a substitution
How to obtain single CSP value from resonances of proton and the attached atom – identify binding surfaces
(1) With a correction for the different spectral widths of e.g. 1HN and 15N:
(2) 𝜕 =√(𝑥(𝜕〖𝐻𝑁〗^2+(𝜕〖𝑁〗^2 )
𝜕 : chemical shift for a particular resonance
x: the number of times of the N dimensional range over that of the H dimensional range, for example, if the range for the N is 30 but 6 for the H , the x should be 5
