Ligands and Interactions Flashcards
What are intermolecular interactions needed for?
They are are essential for cell signalling
Cell signals result from binding e.g. G-protein receptor
The binding initiates a cascade of reactions (often regulated by small molecule regulators)
E.g. Binding of extracellular ligands to receptors to produce an output in the cell
What can we apply intermolecular interactions to?
Intermolecular interactions underpins a lot in the pharmaceutical/biopharma industry
E.g. Imatinib binding to BcrAbl kinase: chronic myeloid leukaemia drug
What are some methods for detecting macromolecular interactions?
Qualitative:
Direct visualisation
Genetic methods (e.g. complementation, yeast two-hybrid system)
Quantative:
Biophysical binding assays
Qualitative & Quantitative:
Biochemical binding assays
Qualitative - whether something is binding or not
Quantitative - how strongly something is binding
Why do we need quantitative methods for detecting macromolecular interactions?
Understand the relationship of the proteins
Understand proportion of complexes at particular concentrations
E.g. Inhibitors for drugs
We can see:
Stable versus transient complexes
Strong versus weak interactions
Type of interactions involved (e.g. hydrophobic, ionic, polar)
Relation between protein/ligand structure and strength of the complex
Understanding of regulation of affinity by posttranslational modification (phosphorylation) and solution conditions (e.g. salt concentrations and ions) - either directly or allosterically
What can be used for direct visualisation?
We use fluorescence microscopy
This is needed to see the interactions to understand the quantitative data
We can add multiple colours to form very useful images - using different fluorophores
E.g. in a dividing cell stain the DNA, centromere and microtubules different colours
We can see localisation within cells and proximity/co-localisation
How does fluorescence work?
A photon is absorbed by a fluorophore and the excited electrons move to a higher energy level (the first excited state) in a transition
When the excited molecule relaxes a new low energy photon is emitted
Measured using a fluorimeter
Commonly measured at 90º or as backscatter because fluorescence is emitted in all directions
The emission spectrum is shifted to a longer wavelength compared with the excitation spectrum (Stokes shift)
What type of fluorescence is measured in direct visualisation?
Extrinsic fluorescence: an extrinsic fluorophore is one added to the macromolecule
It should be attached at a single site and not change its properties
Unlikely it’s intrinsic fluorescence as the macromolecule would need to contain (W, Y or F) - mainly tryptophan as highest yield
What are some issues with direct visualisation?
Optical resolution of the microscope is limited to 200nm
Even with super-resolution in fixed cells 20-100nm (PALM/STORM) a protein or a complex is still limited to 2-20 nm in size i.e. below the resolution limit
Light microscope cannot discern direct from indirect interactions and the fact we see a superposition of the colour, could just mean these components are close in space not necessarily are interacting
What is FRET?
Fluorescence (or Förster) Resonance Energy Transfer:
If the emission of a donor fluorophore overlaps with the absorption peak of a second acceptor fluorophore the energy can be transferred between them in a nonradiative process
It’s like a molecular ‘ruler’ - distance measurement within 2-8 nm
The donor and acceptor are in close spatial proximity
Close proximity can be achieved via a common partner e.g. DNA
FRET doesn’t necessarily imply binding
Can use YFP (yellow) and CFP (cyan) as extrinsic fluorophores (variants of GFP)
What are some biochemical assays used to detect macromolecular interactions?
Co-Immunoprecipitation assay
Pull-down assays
ELISA
Describe the co-immunoprecipitation assay?
- An antibody specific for a target protein is non-covalently linked to a beads coated in protein A or G (proteins of bacterial origin, interact with Fc region on antibody)
- A complex protein mixture is added
- The target protein becomes captured by the specific antibody, remaining bound to its partners
- The bound beads are sepearated by centrifugation, then SDS-PAGE
- The Mr is determined by the gel or westernblotting using antibodies
(A Divan, J Royds, 2013)
What controls should be used for the co-immunoprecipitation assay?
No antibodies present
Use an irrelevant antibody from the same species and form
Use a protein sample with no target protein
(A Divan, J Royds, 2013)
Describe the pull-down assay?
It relies on high affinity attraction
- A recombinant bait protein containing a tag is bound to an affinity resin via the tag
- The prey protein is added and incubated
- This is washed twice with a buffer to remove non-specifically bound and unbound proteins
- It is then eluted by: protease cleavage (between tag and protein) or by disrupting the tag (between resin and tag)
- This undergoes SDS-PAGE, followed by western blotting or mass spectrometry
(A Divan, J Royds, 2013)
What factors should be considered in pull-down assays?
Type of interaction between bait and prey protein:
Obligate - stable interaction
Detected in a range of buffer conditions
Transient - less stable interaction e.g. signalling molecules
Require more defined salt concentrations and pH in a buffer
Require a higher quantity of protein for positive results
Changing elution buffer conditions can tell us about the properties of the protein interaction interface
(A Divan, J Royds, 2013)
What are some fusion tags used in pull down assays?
GST - Glutathione S-transferase
His6 - Hexahistidine
Strep-tag
(A Divan, J Royds, 2013)
What are some problems will the pull-down assays?
Large tags can influence folding/structure or sterically hinder the prey protein binding
If tagged proteins bind to strongly they can be difficult to elute
False-positives with non-specific interactions can occur and nucleic acids frequently co-purify with the protein
(A Divan, J Royds, 2013)
It can take 1-2 hours to complete
You can therefore get dissociation of the complex during washing = negative result when there might have been a complex
Describe the ELISA assay?
Enzyme Linked Immunosorbant assay
- Immobilise the first antibody on a solid support
- Incubate with a protein containing sample
- Add a second antibody that is covalently linked to an assayable enzyme
- Wash and assay enzyme activity
The amount of substrate converted to product indicates amount of protein present
What are the dynamics of intermolecular interactions?
These interactions form an equilibrium
Receptor + Ligand ⇌ Receptor:Ligand complex
Forward reaction - K(on)
Backwards reaction K(off)
Stability of the complex is characterised by the dissociation rate constant K(off) - if dissociation is too fast then pull-down assays will not work
Strength of the interactions is related to the equilibrium dissociation constant Kd
The lower the Kd (dissociation) the stronger the interaction
What is significant in biochemistry about the dynamics of equilibrium?
We use Kd (the dissociation constant) rather than Keq
Kd = 1/Keq
Therefore: ΔG° = RT ln(Kd)
*Notice there isn’t a (-) before RT
The lower the Kd the more negative the (ΔG°) = stronger association interactions, tighter binding
ΔG° decreases by 6 kJ.mol-1 for every 10-fold drop in Kd
What can gibbs free energy be used for in relation to intermolecular interactions?
Free energy is a state function (additive)
This follows Hess’ law, where no matter which path is taken the free energy change is the same
This leads to computationally predicting complex stability by adding contributions from individual interactions, e.g. hydrogen bonds, salt bridges and hydrophobic contacts
This can lead to virtual drug design and screening
How do we determine Kd?
We change the concentration of the ‘free’ ligand and measure the amount of R:L complex bound in a fraction
This will form a rectangular hyperbole
As we add more ligands R:L complexes increase but becomes asymptotic as there is only so many receptors (saturation curve)
Then curve fitting in carried out by software (non-linear regression software e.g. GraphPad)
R0 - amount of receptor (when the graph reaches asymptote)
Kd - concentration of ligand which achieves half saturation of the receptor (fraction bound = 0.5)
How do you determine Kd without computer software?
Convert to a linear format - Scatchard Plot
Bound/Free V Bound is plotted
Slope = -1/Kd
Intercept on the x-axis = Vmax (amount of receptor we have)
How do we meaure the concentration of R:L complex?
- Radiolabel the ligand
- Incubation the ligand with cells
- Wash to remove the majority of the non-specifically bound ligands
- Separation of bound ligand from unbound (by centrifugation, keeping the cell pellet and disgarding the supernatant
- Count the cell fractions and the amount of radioactivity present in the cell = bound label per cell (amount of bound ligand present)
Have a control for nonspecific binding (non-saturable) – competition with excess unlabelled ligand
This needs to be subtracted from the total binding
What should be used as a control for measuring the concentration of R:L complexes?
Have a control for nonspecific binding (non-saturable) – competition with excess unlabelled ligand
This needs to be subtracted from the total binding
If we repeat with a large excess of unlabelled ligand: it will compete out the specific binding but it won’t compete out all the non-specific binding (because there’s so many non-specific binding sites)
