DD - Ligand Binding 1 (C.O.L)

Question 1

What are the 4 main functions manifested in life?

Answer 1

1. Metabolism- The sum of the chemical reactions that occur within a living organism
2. Growth- An increase in dry weight of an organism through cell division and cell enlargement
3. Reproduction- The production of new/similar individuals from parent organisms
4. Response to stimuli- Involves adaptation to the environment

Question 2

What forces are involved in molecular recognition?

Answer 2

Non-covalent forces

Question 3

What are 2 ways a system is characterised?

Answer 3

1. Kinetics- the rate at which something appears or disappears
2. Affinities- the strength of the interaction between macromolecules e.g., PROTEIN + LIGAND

Question 4

What is Kᴀ?

Answer 4

Association contant

Question 5

What is Kᴅ?

Answer 5

Dissociation constant

Question 6

How are Kᴀ and Kᴅ related?

Answer 6

They are inversely related

Question 7

Out of Kᴀ and Kᴅ which one is dimensionless?

Answer 7

Kᴅ is dimensionless
Kᴀ has units kJ/mol

Question 8

What does a low Kᴅ value indicate ab ligand binding?

Answer 8

Ligands bind tightly

Question 9

What does Kᴅ correspond to?

Answer 9

The [Ligand] at which the protein is half saturated

Question 10

What is a binding isotherm?

Answer 10

A plot of the fractional occupancy, f, as a function of [Ligand] at a constant temperature

Question 11

What is the shape of a binding isotherm?

Answer 11

Rectangular hyperbola

Question 12

What does deviation from the shape show?

Answer 12

Shows more complex scenarios
- Multiple binding sites with different affinities or allosteric regulation

Question 13

What are the 4 assumptions inherent in the law of mass action?

Answer 13

1. All receptors are equally accessible to ligands
2. All receptors are either free or bound to ligand (Model ignores partial binding)
3. Neither ligand nor receptor are altered by binding
4. Binding is reversible

Question 14

How are Ligand-Protein complexes measured?

Answer 14

Involves use of binding assays
(Fixed amount of protein is exposed to increasing amounts of ligand)
No further change shows equilibrium

Question 15

How does a Filter binding assay work? (5)

Answer 15

1. Detect binding using radiolabelled ligand
2. Mix with unlabelled ligand
3. Add to sample (of protein) and wait to reach eqm
4. Separate bound ligand and unbound using nitrocellulose filter
5. Total bound is measured using a liquid scintillation counter

Question 16

What also must be measured in binding assays?

Answer 16

Non-specific binding
- Binding to other proteins
- Binding to plastic
Once curve is corrected for non-specific binding Kᴅ can be measured at half saturation