9 - Ligand Binding and Equilibria I

Question 1

What does ligand binding allow for?

Answer 1

Molecular recognition in biology

Question 2

What are some examples of molecular recognition?

Answer 2

Antibodies, DNA and transcription factor, receptors, etc.

Question 3

What is the general equation for ligand binding?

Answer 3

P + L –> PL

Question 4

What type of interaction is ligand binding?

Answer 4

A noncovalent interaction

Question 5

What is the formula for Ka (association constant)?

Answer 5

Ka = [PL]/[P][L]

Question 6

What is the formula for Kd (dissociation constant)?

Answer 6

Kd = [P][L]/[PL]

Question 7

What is the relationship between Kd and Ka?

Answer 7

Kd = 1/Ka

Question 8

What are the units of Kd?

Answer 8

M

Question 9

If a Kd is small, is this a strong or weak association?

Answer 9

Strong association (weak disassociation)

Question 10

If a Kd is large, is this a strong or weak association?

Answer 10

Weak association (strong dissasociation)

Question 11

In a cell, what does Kd reflect?

Answer 11

The concentrations in a cell

Question 12

If DNA concentration is on the order of 1 nM, what is the estimated Kd?

Answer 12

~ 1 nM (same order of magnitude)

Question 13

What is the Kd of enzyme-ATP?

Answer 13

Millimolar to Micromolar

Question 14

What is the Kd of signaling protein to a target?

Answer 14

Micromolar

Question 15

What is the Kd of a sequence-specific recognition of DNA by a transcription factor?

Answer 15

Nanomolar

Question 16

What is the Kd of an inhibitor drug to a protein?

Answer 16

Nanomolar to picomolar

Question 17

What is the Kd of biotin binding to aviding?

Answer 17

Femtomolar (one of the strongest noncovalent interactions)

Question 18

What is specific binding limited by?

Answer 18

The number of available sites

Question 19

What is a hallmark of specific binding interactions?

Answer 19

Saturable binding

Question 20

What does a binding isotherm look like in a linear scale?

Answer 20

Exponential saturation

Question 21

What does a binding isotherm look like in a log scale?

Answer 21

Sigmoidal

Question 22

How can Kd be determined from L concentration?

Answer 22

When [P] = [PL], Kd = [L]

Question 23

What is [L]?

Answer 23

Concentration of FREE ligand

Question 24

What is [P]?

Answer 24

Concentration of FREE protein

Question 25

What is [PL]?

Answer 25

Concentration of PL complex

Question 26

What is [L]T?

Answer 26

Total ligand concentration ([L]T = [L] + [PL])

Question 27

What is [P]T?

Answer 27

Total protein concentration ([P]T = [P] + [PL])

Question 28

In an experiment, what quantities in Kd are usually known?

Answer 28

[P]T and [L]T (NOT [L] and [P])

Question 29

What happens if [P]T < Kd?

Answer 29

There is very little [PL], so [L] = [L]T

Question 30

What happens if [P]T > Kd?

Answer 30

A lot of complex [PL] is formed, so [L] < [L]T

Question 31

What is f?

Answer 31

Fractional occupancy

Question 32

What is the equation for f?

Answer 32

f = [PL]/([PL}+[P])

Question 33

What happens at f = 0.5?

Answer 33

Kd = [L]

Question 34

If a binding isotherm plots f vs. [L], how can Kd be determined?

Answer 34

The [L] where f = 0.5

Question 35

If a binding isotherm plots f and [L]T, how can you determine Kd?

Answer 35

Need more complicated math (Kd does not equal [L])

Question 36

What is the challenge with plotting f with [L]?

Answer 36

[L] is usually very hard to determine, while [L]T is very easy to determine

Question 37

How can f be experimentally calculated?

Answer 37

By plotting [PL] vs [L], and seeing where the curve saturates

Question 38

In a [PL] vs. [L] isotherm, how can Kd be determined?

Answer 38

By finding the half maximal point (f = 0.5), and Kd = [L] at this point

Question 39

What thermodynamic quantity changes upon ligand binding?

Answer 39

Delta G

Question 40

What is delta G bind?

Answer 40

The energy released when binding (P + L --> PL)

Question 41

What is the formula for delta G bind?

Answer 41

Delta G bind = RT ln(Kd)

Question 42

If a Kd is small, what can you say about delta G bind?

Answer 42

It is more negative (higher magnitude)

Question 43

If a Kd is large, what can you say about the delta G bind?

Answer 43

It is less negative (lower magnitude)

Question 44

What is the formula for delta G?

Answer 44

Delta G = delta H - T delta S

Question 45

What does delta H come from in ligand binding?

Answer 45

Hydrogen bonds (distance dependent, geometric constraint), electrostatic bonds (Coulomb's law), salt bridges

Question 46

How does ligand binding decrease entropy?

Answer 46

There is less degrees of freedom, hydrophobic effect

Question 47

How does ligand binding increase entropy?

Answer 47

When ligand binds, water is released, which increases entropy (same if ligand is solvated)

Question 48

In delta G, what is easy to predict and why?

Answer 48

H, because you can estimate based on the bonds and energetics

Question 49

In delta G, what is hard to predict and why?

Answer 49

S, because there are compensating effects

Question 50

How does the filter binding assay work?

Answer 50

Two test tubes with [P] and [L] are mixed, and the [L] is separated from the [PL] and [P] using the filter

Question 51

How is Kd determined from a filter binding assay?

Answer 51

Plot f and L to find Kd

Question 52

How does fluorescence polarization / anisotropy work?

Answer 52

A fluorophore is used to measure tumbling. There is less tumbling when bound, so changes in tumbling can be plotted

Question 53

How is Kd determined from fluorescence polarization / anisotropy?

Answer 53

Can plot tumbling changes, and fit to a Kd

Question 54

How does FRET work?

Answer 54

Direct observation of two probes (see changes in concentration)

Question 55

How does the bead halo assay work?

Answer 55

A halo can be detected through microscopy, so small changes in fluorescence can be measured

Question 56

What are some methods to measure Kd?

Answer 56

1. Filter binding assay 2. Fluorescence polarization / anisotropy 3. FRET 4. Bead halo assay 5. Isothermal titration calorimetry 6. Surface plasma resonance 7. Biolayer interferometry 8. Gel shift 9. Co-sedimentation (SEC, AUC)

Question 57

What is the general method for ligand binding assays?

Answer 57

Detect changes of the system due to ligand binding (shape changes, etc.)

Question 58

What concentration ranges are needed for a Kd assay?

Answer 58

A concentration range where Kd is in the middle

Question 59

What is the purpose of saturation measurements?

Answer 59

Bound fraction remains small

Question 60

Why is radioactivity used in ligand binding?

Answer 60

Only way to not change chemistry of ligand

Question 61

How is retinoic acid used in ligand binding?

Answer 61

Radioactive retinoic acid is combined with the receptor, and charcoal is used to separate free ligand

Question 62

What is the purpose of radioactivity with retinoic acid?

Answer 62

Calculate radioactivity to measure free ligand

Question 63

What assumption is used in the retinoic acid test?

Answer 63

Equilibrium time is longer than separation time (experiment is very specific to retinoic acid)

Question 64

What is retinoic acid?

Answer 64

Metabolite of vitamin A

Question 65

When is the Scatchard analysis done?

Answer 65

Estimate Kd when receptor concentration is unknown

Question 66

How does the Scratchard analysis work?

Answer 66

Plot [Lbound]/[L] vs. [Lbound] to find Kd and [P]T

Question 67

What is the formula for Scatchard analysis?

Answer 67

[Lbound]/[L] = (-1/Kd)[Lbound] + [P]T/Kd

Question 68

What is the slope of Scatchard analysis?

Answer 68

-1/Kd

Question 69

What is the x intercept of a Scatchard analysis?

Answer 69

[P]T / Kd

Question 70

What does the Hill equation describe?

Answer 70

The binding isotherm

Question 71

What is the Hill equation?

Answer 71

f^n = L^n / (L^n + Kd)

Question 72

What is n?

Answer 72

Hill coefficient

Question 73

What happens when n = 1?

Answer 73

No cooperativity

Question 74

What happens when n < 1?

Answer 74

Negative cooperativity

Question 75

What happens when n > 1?

Answer 75

Positive cooperativity

Question 76

Where does the biggest change of f occur?

Answer 76

Between 0.1 and 10 of L/Kd

Question 77

What is the chemical equation for competition?

Answer 77

PA + L P + L + A PL + A (two Kd)

Question 78

What does the Cheng-Prusoff equation describe?

Answer 78

Relate IC50% to Ki (more standardized value)

Question 79

What does ITC stand for?

Answer 79

Isothermal titration calorimetry

Question 80

What is ITC used for?

Answer 80

Measuring thermodynamics of binding?

Question 81

How does ITC work?

Answer 81

Drops of ligand are added, and the heat released is measured, as well as the binding isotherm

Question 82

How does ITC measure delta G?

Answer 82

It uses the binding isotherm to find Kd, which can be used to calculate delta G

Question 83

What is the formula for ITC Kd?

Answer 83

f = (L/Kd) / (1 + L/Kd)

Question 84

How does ITC measure delta H?

Answer 84

It uses the heat released

Question 85

How doe ITC measure delta S?

Answer 85

It uses delta G and delta H to calculate delta S

Question 86

What is lock and key binding?

Answer 86

No change in conformation

Question 87

What is induced fit binding?

Answer 87

There is a conformational change in the protein

Question 88

What is the consequence of a protein interconverting between many states?

Answer 88

Binding of a ligand can select one state over another

Question 89

What is ligand induced folding?

Answer 89

Binding of a ligand changes the protein shape

Question 90

What is conformational selection?

Answer 90

A ligand binds in only one state, so it selects the particular state

Question 91

True or false: the timescale of protein conformational dynamics affects binding

Answer 91

True: this determines how often a state is available to be selected for by the ligand

Question 92

What is the Cheng Prusoff equation?

Answer 92

Ki = IC50% / (1 + ([A]/Kd2))