Ligands and Interactions

Question 1

What are intermolecular interactions needed for?

Answer 1

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

Question 2

What can we apply intermolecular interactions to?

Answer 2

Intermolecular interactions underpins a lot in the pharmaceutical/biopharma industry
E.g. Imatinib binding to BcrAbl kinase: chronic myeloid leukaemia drug

Question 3

What are some methods for detecting macromolecular interactions?

Answer 3

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

Question 4

Why do we need quantitative methods for detecting macromolecular interactions?

Answer 4

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

Question 5

What can be used for direct visualisation?

Answer 5

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

Question 6

How does fluorescence work?

Answer 6

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)

Question 7

What type of fluorescence is measured in direct visualisation?

Answer 7

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

Question 8

What are some issues with direct visualisation?

Answer 8

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

Question 9

What is FRET?

Answer 9

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)

Question 10

What are some biochemical assays used to detect macromolecular interactions?

Answer 10

Co-Immunoprecipitation assay
Pull-down assays
ELISA

Question 11

Describe the co-immunoprecipitation assay?

Answer 11

1. 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)
2. A complex protein mixture is added
3. The target protein becomes captured by the specific antibody, remaining bound to its partners
4. The bound beads are sepearated by centrifugation, then SDS-PAGE
5. The Mr is determined by the gel or westernblotting using antibodies

(A Divan, J Royds, 2013)

Question 12

What controls should be used for the co-immunoprecipitation assay?

Answer 12

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)

Question 13

Describe the pull-down assay?

Answer 13

It relies on high affinity attraction

1. A recombinant bait protein containing a tag is bound to an affinity resin via the tag
2. The prey protein is added and incubated
3. This is washed twice with a buffer to remove non-specifically bound and unbound proteins
4. It is then eluted by: protease cleavage (between tag and protein) or by disrupting the tag (between resin and tag)
5. This undergoes SDS-PAGE, followed by western blotting or mass spectrometry

(A Divan, J Royds, 2013)

Question 14

What factors should be considered in pull-down assays?

Answer 14

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)

Question 15

What are some fusion tags used in pull down assays?

Answer 15

GST - Glutathione S-transferase
His6 - Hexahistidine
Strep-tag

(A Divan, J Royds, 2013)

Question 16

What are some problems will the pull-down assays?

Answer 16

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

Question 17

Describe the ELISA assay?

Answer 17

Enzyme Linked Immunosorbant assay

1. Immobilise the first antibody on a solid support
2. Incubate with a protein containing sample
3. Add a second antibody that is covalently linked to an assayable enzyme
4. Wash and assay enzyme activity

The amount of substrate converted to product indicates amount of protein present

Question 18

What are the dynamics of intermolecular interactions?

Answer 18

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

Question 19

What is significant in biochemistry about the dynamics of equilibrium?

Answer 19

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

Question 20

What can gibbs free energy be used for in relation to intermolecular interactions?

Answer 20

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

Question 21

How do we determine Kd?

Answer 21

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)

Question 22

How do you determine Kd without computer software?

Answer 22

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)

Question 23

How do we meaure the concentration of R:L complex?

Answer 23

1. Radiolabel the ligand
2. Incubation the ligand with cells
3. Wash to remove the majority of the non-specifically bound ligands
4. Separation of bound ligand from unbound (by centrifugation, keeping the cell pellet and disgarding the supernatant
5. 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

Question 24

What should be used as a control for measuring the concentration of R:L complexes?

Answer 24

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)

Question 25

What are some limitations of the simple binding assay for measuring the amount of R:L complexes?

Answer 25

Stability of the complex during washes - if Kd > 100 nM then we have loss of bound ligand
Detection and separation of bound ligand may be difficult
Non-specific binding may completely mask specific signal
Limited to low receptor concentrations – we need strong binding and strong signal to be detected

Question 26

What happens if there is low signal/low affinity?

Answer 26

The receptor concentration may need to be higher and is no longer negligible
Therefore we need to solve the full set of equations (‘tight binding’ regime)
Subsitute into a quadratic equation - called Morrison equation

Question 27

Describe a low signal/affinity that requires a high receptor concentration?

Answer 27

HABA - a biotin analogue
Through absorbance we can see HABA binding to avidin
The Kapp is low - 5.94 µM
True Kd = 4 µM
Therefore Kd is overestimated – unless we use the ‘tight binding’ equation - Morrison equation

Question 28

What is a competition assay?

Answer 28

A different ligand that competes for a binding site
L + R -> L:R _ + C -> C:R +L
L = labelled
C = competing ligand

This assay is useful for drug discovery as we can see a drug competing to bind with a substrate for a certain binding site
We are interested in competitor dissociation constant = Kc
L:R is measured as C isn’t labelled

Question 29

What are the Kapp equations?

Answer 29

Kapp = apparent dissociation constant
Kapp = Kc(1+[L]/Kd)
Therefore
Kc = Kapp/(1+[L]/Kd)

Kapp often referred to as the IC50
Kc is also often referred to as Ki

If we can measure Kapp - we measure the mid point of the curve due to different affinities for the receptor - they are different = defining point

Question 30

What is the competition assay when [L] = Kd

Answer 30

They are often run at this parameter where the receptor is half-saturated with labelled ligand

If you put the numbers into Kc = Kapp/(1+[L]/Kd)
Kapp = 2 x Kc
OR
IC50 = 2 x Ki

Question 31

What is isothermal titration calorimetry?

Answer 31

This measures precise ΔH directly from the heat released or absorbed
We can do a label-free quantitative binding assay
From the concentration-dependence we get Ka

We can work out: ΔG = -RT ln(Ka) and therefore ΔS

From a complete thermodynamic description we can inform computer-based (in silico) methods
Mainly used for characterisation of drugs not necessarily screening as there isn’t the highest throughput

Question 32

What are the kinetics of binding?

Answer 32

Strong binding = favourable interaction = spontaneous reaction upon mixing
During binding entropy usually decreases* ΔS <0 (however, consider hydrophobic effect)
ΔH < 0 heat is released = exothermic reaction

Question 33

What happens within the Isothermal titration calorimetry instument?

Answer 33

There’s a sample cell just buffer) and reference cell - it measures ΔT between the two cells
For this experiment we get a spike for a release of heat for each addition of ligand, until it dwindles off
We can then plot kcal/mol of injectant per molar ratio - sigmodal binding curve

Sensitive feedback heating loop for µJ heat measurements

Question 34

What do we need to be aware off with the ΔH value?

Answer 34

ΔH depends on temperature

The heat capacity also changes with temperature
Therefore you need to record at what temperature you are taking the reading at

Question 35

What are the limits of isothermal titrational calorimetry?

Answer 35

Sensitivity of the instrument sets the lowest amount of added ligand and protein that can be used to obtain signal above noise
This is very sensitive

At high affinity all the added ligand is bound till the protein is exhausted – steep concentration dependence – importance of the ‘C-value’ - ratio of Kd to the protein concentration

Question 36

Describe rate of biochemical reactions?

Answer 36

Reaction rate is governed by a rate constant
Simple measurement over time = kinetics
We measure the binding over time

Rate = k[A][B]

Question 37

What is the reaction order?

Answer 37

Reaction order = Number of molecules needed to collide and form the activated complex
The initial rate depends on the reactant concentration

Zero
First - linear
Second - quadratic

Ligand binding kinetics is almost always measured under pseudo-first order conditions (keep one concentration the same)

Question 38

What is the rate order with reversible reactions?

Answer 38

First order reversible reactions = AP
Forward rate constant kf, reverse kr
Kapp=(kf + kr) - the sum of the forward and backwards rate constants