Ligand Binding Flashcards
What are the units of the association constant KA?
M-1
What are the units of the dissociation constant KD?
M
What are the units of on rate constant?
M-1s-1
What are the units of off rate constant, kD?
What is the relationship between KD and KA?
KD= 1/KA
What’s the formula for association constant?
KA=kA/kD
What’s the formula for dissociation constant?
KD=kD/kA
What is the formula for calculating ΔGº from KA?
ΔGº= -RTlnKA
What is the formula for calculating ΔGº from KD?
ΔGº=+RTlnKD
What is a typical KD value for enzyme-substrate interactions?
1 x 10-3 to 1 x 10-6
What is a typical KD value for a signalling protein binding to its target?
1 x 10-6
What is a typical KD value for small molecule inhibitors of proteins (drugs)?
1 x 10-9 to 1 x 10-12
What is the strongest known non-covalent interaction?
Biotinbinding to adivin
What is KD?
The concentration of free ligand at which 50% of the protein is saturated
How can we determine the value of KD through binding assays?
- Measure [P•L} using absorbance or fluorescence
- Using radiolabelled ligand to measure [P•L}
How can [P•L} be measured using absorbance or fluorescence?
- A fluorescent tag is put onto the ligand
- The tag shows low fluroscence when unbound
- When bound the tag shows high fluorescence
How can [P•L} be measured using radiolabelled ligand?
- Solution of ligand with radioactivity is added to a solution of receptor
- A resin which binds to the receptor protein is then added
- The mixture is then centrifuged and all receptor proteins are at the bottom of the tube bound to the resin
- The supernantant is discarded and radioactivity is measured
When do weak binding conditions occur?
When [L]free ≈ [L}bound
What is the formula for fractional binding under weak binding conditions?
f= [L}total/KD + [L]total
When do tight binding conditions occur?
When [L]free =/= [L]total
What do tight binding conditions allow to be determined about the reaction?
Allows the stoichiometry of the protein-ligand interaction to be determined
What equation is used in a scatchard analysis?
[L]bound/[L]= (-1/KD) x [L]bound + ([P]total/KD)
What direction does a scatchard analysis go?
Downhill due to the negative gradient
Outline how to complete a ligand binding experiment for a scatchard analysis
- Solution of receptor is added to ligand spiked with radioctivity
- Charcoal pellet are added
- Mixture is centrifuged and charcoal pellet absorbs any unbound ligand
- Pellet is removed leaving receptors and bound ligand
What is on the x axis of a scatchard plot?
[L]bound
What is on the y axis of a scatchard plot?
[L]bound/ [L]free
What is Ki?
The dissociation constant for inhibitor
A higher concentration of drug causes a rate-substrate graph to move in which direction?
To the right
What is allostery?
The communication between 2 sites in a protein
What is homotropic allostery?
Where the regulating ligand is a substrate
What is heteotropic allostery?
When the regulating ligand binds at a distance from the active site
What is an advantage of allostery?
It allows for proteins to be ultrasensitive to their environments
Does a cooperative response require allostery?
No but often involves it
Give an example of a chemical which allosterically regulates glycolysis
Phosphofructokinase
