Protein regulation 1: binding and enzyme kinetics

Question 1

What is KD?

Answer 1

Equilibrium dissociation constant; the concentration of ligand at 50% maximal binding

measure of affinity

Question 2

Tight complex vs weak complex KD? Gibbs free energy??

Answer 2

Weak complex = larger KD (mM)
- small delta G

Tight complex = smaller KD (nM)
- large delta G

Question 3

Delta G equation?

Answer 3

Delta G = -RTln(KD)

Question 4

KD equation?

Answer 4

[P].[L]/[PL]

conc protein x conc ligand / conc complex

Question 5

2 ways of measuring KD?

Answer 5

Equilibrium methods; measure concs. of ligand + protein + complex
e.g. equilibrium dialysis, titration calorimetry

Kinetic methods; measure rates to determine rate constants
e.g. Rapid mixing techniques (stopped-flow fluorescence, surface plasmon resonance)

Question 6

Cons of equilibrium dialysis? (aside from its old and bad)

Answer 6

Requires accurate measurement of [L]
Needs lots of protein
Time needed to reach equilibrium 
Sticking of protein to dialysis tube
Cumbersome (esp. with large nos. samples)
Only good for narrow range of KDs