Lecture 4 Molecular recognition

Question 1

Allosteric interactions

Answer 1

When the binding of a molecule alters the affinity of the other molecules for the receptor.

Question 2

Association constant, Ka

Answer 2

Ka = [P x L] / [P][L]
[P]: concentration free protein
[L]: concentration of free ligand

Question 3

Specificity of the interaction

Answer 3

relative strenght of the interactions made between one protein and alternative ligand.
Biologically relevant interactions are usually highly specific

Question 4

Dissosiation constant Kd

Answer 4

1/Ka

Question 5

Fractional saturation

Answer 5

The extent to which the binding site on a protein are filled with ligand.
f = [L] / ([L] + Kd)
when f = 0,5 have of the protein have bound ligand

Question 6

Why is KD used more often than KA?

Answer 6

The value of KD is equal in magnitude to the concentration of free ligand at which half of the protein molecules are bound to ligand and (and half are unliganded) at equilibrium.

Question 7

Estrogen receptors

Answer 7

When estrogen binds to the receptor it promotes the dimerization of the receptor, which facilitates the binding of the receptor to sites on DNA that contain specific recognition sequences. Binding of estrogen to the receptor also induces a conformational change in the ligand binding domain, which results in the recruitment of proteins known as transcriptional co-activators to the receptor. The co-activator protein are responsible for turning on transcription from the gene.

Question 8

Measuring estrogen binding

Answer 8

1. Solution of estrogen contains a known fraction of radioactively labelled estrogen.
2. Negatively charged resin is added to the solution.
   3.The estrogen receptor binds to the resin.
3. Configuration step separates the bound from the unbound ligand.
   5.The pelleted resin with the protein is transferred to a vial and the total amount of radioactivity in it is estimated by using a liquid scintillation counter.

Question 9

Scatchard analysis

Answer 9

Involves rearranging the basic binding equation.
It is an alternative form of the hyperbolic binding equation and tells us that the ratio of bound to free ligand concentration is related linearly to the bound ligand concentration.

Question 10

Scatchard analysis for retinoic acid receptor

Answer 10

1. Cells expressing the receptor are lysed and the cell lysate containing the receptor is used for a series of binding experiments.
2. Samples containing normal retinoic acid are spiked with retinoic acid that contains the radioactive isotope tritium.
3. For each binding measurement, the receptor containing lysate and the radioactive labelled retinoic acid are mixed together.
4. Pellets of charcoal are added to the reaction mixture, the free retinoic acid binds to it (bound to receptor it doesn’t) and the bound charcoal is separated from the solution by centrifugation.
5. Radiation in bound and unbound retinoic acid can now be measured.

Question 11

Saturable binding

Answer 11

When a ligand binds to a single binding site on a protein and no other, app of the protein molecules are bound to ligand at very high ligand concentration. Increasing the ligand concentration beyond this point does not lead to any increase in protein binding. -> platform in the isotherm
When a binding isotherm does not saturate, even at very high ligand concentrations, then it usually indicates that the ligand is also binding to other things than the target protein.

Question 12

Tight interaction

Answer 12

KD as low as possible.

Question 13

Lead compound

Answer 13

Small molecules that inhibit a target protein but do not have high affinity or other desirable properties. Drug development often involves the systematic modification and optimization of lead compounds

Question 14

Protein kinases

Answer 14

Enzymes that transfer the terminal phosphate of ATP to the hydroxyl groups of serine, threonine, or tyrosine residues on proteins.

Question 15

Targets in HIV treatment

Answer 15

HIV reverse transcriptase: nucleotide analogs
HIV protease: protease inhibitors

Question 16

Conformational selection

Answer 16

Induced fit binding is thought to involve the bonding of a ligand to conformations of the receptor that are populated although at a low level, even in the absence of ligand

Question 17

Induced fit

Answer 17

Model for binding of a ligand to a receptor, the binding site is considered to have some plasticity, allowing it to change somewhat to accommodate binding of the ligand or substrate, like a hand fitting into a stretchy glove.

Question 18

Statins

Answer 18

a class os small molecule drugs that are effective in lowering cholesterol levels in humans. They inhibit an enzyme known as HMG-CoA reductase, which catalyzes a key step in the synthesis of cholesterol.

Question 19

IC50 value for an inhibitosr

Answer 19

The concentration of inhibitor that reduces the activity of a protein to half the maximal value