Protein Function Flashcards
How is the affinity of an interaction determined? What is actually being measured?
Affinity determined by the ratio of bound ligand to unbound ligand at equilibrium
- protein has HIGH affinity to ligand (incr specific rxns, on-rate > off-rate)
- protein has LOW affinity to ligand (decr specific rxns, off-rate > on-rate)
How are Ka and Kd defined? What do these terms imply about any interaction between a protein and its ligand?
Ka = binding equilibrium/association constant
Kd = dissociation constant, ligand concentration when 50% of maximal binding is achieved (θ = 0.5 on curve)
incr Ka = decr Kd
If protein has high affinity for ligand, Ka should be high and Kd low
Curve doesn’t hit 100% (1.0) b/c off-rate exists, 1% is always releasing
Cooperativity
Binding of one molecule of ligand/substrate incr affinity of binding for addnl molecules at other sites
Small changes in ligand suddenly produce large cooperative effects on binding
What drives cooperative binding of O2 in hemoglobin?
Binding of first O2 molecule = low affinity
Binding of first alters hemoglobin’s structure, now other hemes have higher affinity
What are the T and R states of hemoglobin? How do they contribute to cooperativity?
T (tense) state = lower affinity (high Kd)
R (relaxed) state = higher affinity (low Kd)
Binding of O2 to heme drives partial relaxation of adjacent heme, opening up next heme for higher affinity
Continued relaxation as more binding occurs
Why is cooperative binding of O2 important for Hemoglobin’s function in O2 transport from the lungs to peripheral tissues?
Hemoglobin senses higher O2 in lungs - cooperatively maximizes O2 binding to be carried away
Hemoglobin senses lower O2 in tissues - changes affinity from R to T (decr affinity), releases O2
Allostery
Binding of a molecule at site A affects the conformation of the protein at distant site B
What is the relationship between cooperative binding and allostery?
Cooperative binding = type of allostery, both involve one site affecting another indirectly
How can ATP hydrolysis be coupled to protein conformational changes to provide work?
ATP hydrolysis (expansion), product incr in size*
What “reaction” is being energetically coupled to ATP hydrolysis in muscle contraction?
Rxn w/ myosin
What would happen to contraction if ATP hydrolysis was blocked, but ATP binding was not?
Myosin arm wouldn’t be oriented correctly - ATP would be stuck to myosin and energy wouldn’t be “released” for use, myosin head wouldn’t be able to be used for the next round of contraction
Why are ADP and Pi release equally as important for muscle contraction as ATP hydrolysis?
ADP and Pi release replenishes used ATP
What would happen if Pi release was blocked?
The myosin arm wouldn’t have the force pushing it into a specific conformation
How do enzymes increase the rate of catalysis? What is their effect on ΔG?
Incr activation energy, no effect on ΔG
How does affinity to substrate affect the reaction?
Higher affinity allows more change to occur - changes needed to drive catalysis (stretching bonds, exposing sites of attack,
