FOM 1.3.1

Question 1

What is an apo-protein?

Answer 1

protein not bound to anything

Question 2

What is a ligand?

Answer 2

Anything that binds a protein.

Question 3

What is a holo-protein?

Answer 3

A complex of all components (proteins, subunits, and all ligands) needed for function

Question 4

Give examples of some of the things that proteins bind.

Answer 4

small sugars, nucleotides, RNA, water, organic molecules, O2, large carbs, metal ions, proteins, lipids, CO2, protons, DNA, drugs, photons, NO

Question 5

What constitutes a protein binding site?

Answer 5

A well-defined region of the protein that contacts a ligand. These contacts are made by specific AA side chains or backbone.

Question 6

Give an example of a protein binding site interaction with a ligand.

Answer 6

An aromatic tryptophan ring stacking against the hexose ring of the sugar ligand.

Question 7

How does ATP show chemical complementarity?

Answer 7

ATP has numerous domains that interact with binding sites including: Hydrophobic regions, positive charges, accepting and donating H bonds.

Question 8

What happens when an ATP tries to bind in an inverted manner?

Answer 8

Chemical repulsion and steric clashes. ATP won’t bind

Question 9

How can mutations alter binding? (2)

Answer 9

It can affect how tightly the ligand is bound (affinitiy) It can affect which ligand is bound most tightly (specificity, similar to selectivity)

Question 10

What has occurred in patients who present with Vitamin D resistant rickets?

Answer 10

A mutation has occurred in the receptor that alters binding stability b/t receptor and ligand.

Question 11

In the example with Vitamin D resistant rickets, how were doctors able to increase vitamin D uptake by cell lines? What’s the limitation or obstacle with this route?

Answer 11

The doctors were able to alter the vitamin D to better fit w/ the cells mutant receptor. The obstacle is that patients may have different mutations.

Question 12

Can ligands affect the protein structure?

Answer 12

Yes and they most likely will.

Question 13

How do ligands affect the equilibrium of proteins b/t their folded and unfolded form?

Answer 13

Shift equilibrium in favor of the folded forms.

Question 14

What type of roles can metal ions play in proteins? Would we use drugs to mimic these effects?

Answer 14

“structural” roles Yes, drugs that mimic these effects are used in unfolding dz’s (e.g. CFTR)

Question 15

Can different ligands affect the way a protein will fold? If so, how much of a folding difference could be achieved?

Answer 15

Yes 1000-fold difference

Question 16

P + L PL , give the dissociation and association reaction contants, k

Answer 16

Ka = [PL]/[P][L], units M-1 Kd = [P][L]/[PL], units M

Question 17

In the dissociation reaction, when [P]free = [PL], what is Kd equal to?

Answer 17

[L]

Question 18

Where can Kd be found on a sigmoidal curve?

Answer 18

At the 50/50 point, the point where half of the protein is bound.

Question 19

When looking at differentiation curves, what is important to note about the axises? How could it affect the curve?

Answer 19

Standard unit distribution = hyperbolic shape Logarithmic distribution= sigmoidal curve

Question 20

What is the slope around the half-way point of an uninfluenced sigmoidal curve?

Answer 20

1

Question 21

When looking at a single ligand-protein binding sigmoidal dissociation curve, what do shifts to the left (smaller []) and right indicate?

Answer 21

Left: Tighter affinity Right: Weaker affinity

Question 22

When analyzing the capability of a drug to bind a protein, what type of concentration is desirable?

Answer 22

A drug that can achieve a concentration higher than its midpoint inside a cell is desirable.

Question 23

What is the midpoint of a drug concentration in vivo called?

Answer 23

EC50 instead of Keq

Question 24

What type relationship exists when the binding of the first has no effect on the binding of the subsequent?

Answer 24

Independent

Question 25

What two types of relationships are possible if binding the 1st alters binding of the 2nd?

Answer 25

Cooperative, Allosteric

Question 26

The thermodynamic cycle is…

Answer 26

pathway independent

Question 27

Name this type of binding relationship: Same binding site, same ligand

Answer 27

Cooperative

Question 28

Name this type of binding relationship: Different binding site, different ligand

Answer 28

Allosteric

Question 29

Name this type of binding relationship: Different binding site, different ligand

Answer 29

Allosteric

Question 30

Break down the word allostery. Allos: ____ Steric: ____

Answer 30

Allos: other Steric: placement of atoms

Question 31

Give an example of an allosteric interaction

Answer 31

A protein binding w/ DNA & sugar

Question 32

In inhibitory allostery, how is the sigmoidal curve altered?

Answer 32

Shifted to the right b/c its weaker

Question 33

In enhancing allostery, how is the binding sigmoidal curve shifted?

Answer 33

To the left, b/c its stronger

Question 34

What are some possible benefits/uses of allostery in nature?

Answer 34

Regulation! Modulating function instead of on/off Received signal and transform response

Question 35

Give 2 examples of how allostery is utilized w/in the body.

Answer 35

Metabolism - change nutrients to allow more or less metabolism DNA Txn - change transcriptome/proteome in response to environment

Question 36

Give two examples of how cooperativity could be arranged.

Answer 36

Binding repeated subunits on homo-oligomers Multiple identical binding domains on a single polypeptide chain (e.g. calmodulin)

Question 37

The sigmoidal curves represents what of the multiple ligands

Answer 37

weighted average b/c its impossible to distinguish b/t the 2(+) binding sites

Question 38

What happens to the curve with positive cooperativity?

Answer 38

The sigmoidal curve becomes steeper

Question 39

What is different about a linear plot of ligand-protein binding with positive cooperativity?

Answer 39

The positive cooperativity curve shows sigmoidal characteristics

Question 40

What is binding that one ligand makes subsequent binding more difficult?

Answer 40

Negative cooperativity

Question 41

With negative cooperativity, will it ever reach 100% bound?

Answer 41

No

Question 42

What are the 3 ultimate results of positive cooperativity?

Answer 42

Response range to ligand change narrows Dramatic biological effects Closer to on/off

Question 43

What are the 2 ultimate results of negative cooperativity?

Answer 43

Changes @ higher ligand [] don/t affect protein function Constant protein function across a wide range of ligand []s

Question 44

Name at least 3 of the possible advantages of allosteric drugs.

Answer 44

-More subtle effects -Slow function/Ramp up function -Expands # of search targets

Question 45

What are the units for the association reaction? What are the units for the dissciation reaction?

Answer 45

Association M^-1

Dissociation M

Question 46

What is the difference between a sigmoidal and hyperbolic graph?

Answer 46

The hyperbolic graph “tends” to be a classical representation and the sigmoidal graph is in logarithmic function.

Question 47

What is the shifting in this graph demonstrating?

Answer 47

Shift to the right is a weaker affinity and a shift left is a stronger affininty

Question 48

Answer this

Answer 48

B - because it uses the least amount of ligand to bind the most amount of protein. MDV3100 would be the most effective binder.

Question 49

These two graphs demonstrate an example of what?

Answer 49

Inihibitory allostery

Question 50

This graph demonstrates what type of allostery?

Answer 50

Enhancing allostery

Question 51

Explain what each line is and what is happening to the slope of the green line

Answer 51

Pink is pos cooperativity

Orange is no cooperativity

Green is negative cooperativity, and it will never reach 100% of the positive

Question 52

Answer 52

D - The shift to the right shows that there is a higher amount of Ca2+ needed to bind that same amount of CaM. This shows that it is diminishing the binding of Ca2+. The slope of the line is unchanged, meaning that it is allosterically diminishing the effect and not cooperatively.