Ligand Binding

Question 1

What are the units of the association constant K_A?

Answer 1

M^-1

Question 2

What are the units of the dissociation constant K_D?

Answer 2

M

Question 3

What are the units of on rate constant?

Answer 3

M^-1s^-1

Question 4

What are the units of off rate constant, k_D?

Question 5

What is the relationship between K_D and K_A?

Answer 5

K_D= 1/K_A

Question 6

What’s the formula for association constant?

Answer 6

K_A=k_A/k_D

Question 7

What’s the formula for dissociation constant?

Answer 7

K_D=k_D/k_A

Question 8

What is the formula for calculating ΔGº from K_A?

Answer 8

ΔGº= -RTlnK_A

Question 9

What is the formula for calculating ΔGº from K_D?

Answer 9

ΔGº=+RTlnK_D

Question 10

What is a typical K_D value for enzyme-substrate interactions?

Answer 10

1 x 10^-3 to 1 x 10^-6

Question 11

What is a typical K_D value for a signalling protein binding to its target?

Answer 11

1 x 10^-6

Question 12

What is a typical K_D value for small molecule inhibitors of proteins (drugs)?

Answer 12

1 x 10^-9 to 1 x 10^-12

Question 13

What is the strongest known non-covalent interaction?

Answer 13

Biotinbinding to adivin

Question 14

What is K_D?

Answer 14

The concentration of free ligand at which 50% of the protein is saturated

Question 15

How can we determine the value of KD through binding assays?

Answer 15

• Measure [P•L} using absorbance or fluorescence
• Using radiolabelled ligand to measure [P•L}

Question 16

How can [P•L} be measured using absorbance or fluorescence?

Answer 16

• A fluorescent tag is put onto the ligand
• The tag shows low fluroscence when unbound
• When bound the tag shows high fluorescence

Question 17

How can [P•L} be measured using radiolabelled ligand?

Answer 17

• 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

Question 18

When do weak binding conditions occur?

Answer 18

When [L]_free ≈ [L}_bound

Question 19

What is the formula for fractional binding under weak binding conditions?

Answer 19

f= [L}_total/K_D + [L]_total

Question 20

When do tight binding conditions occur?

Answer 20

When [L]_free =/= [L]_total

Question 21

What do tight binding conditions allow to be determined about the reaction?

Answer 21

Allows the stoichiometry of the protein-ligand interaction to be determined

Question 22

What equation is used in a scatchard analysis?

Answer 22

[L]bound/[L]= (-1/K_D) x [L]_bound + ([P]_total/K_D)

Question 23

What direction does a scatchard analysis go?

Answer 23

Downhill due to the negative gradient

Question 24

Outline how to complete a ligand binding experiment for a scatchard analysis

Answer 24

• 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

Question 25

What is on the x axis of a scatchard plot?

Answer 25

[L]_bound

Question 26

What is on the y axis of a scatchard plot?

Answer 26

[L]_bound/ [L]free

Question 27

What is Ki?

Answer 27

The dissociation constant for inhibitor

Question 28

A higher concentration of drug causes a rate-substrate graph to move in which direction?

Answer 28

To the right

Question 29

What is allostery?

Answer 29

The communication between 2 sites in a protein

Question 30

What is homotropic allostery?

Answer 30

Where the regulating ligand is a substrate

Question 31

What is heteotropic allostery?

Answer 31

When the regulating ligand binds at a distance from the active site

Question 32

What is an advantage of allostery?

Answer 32

It allows for proteins to be ultrasensitive to their environments

Question 33

Does a cooperative response require allostery?

Answer 33

No but often involves it

Question 34

Give an example of a chemical which allosterically regulates glycolysis

Answer 34

Phosphofructokinase