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CHE 541 > 9 - Ligand Binding and Equilibria I > Flashcards

9 - Ligand Binding and Equilibria I Flashcards

1
Q

What does ligand binding allow for?

A

Molecular recognition in biology

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2
Q

What are some examples of molecular recognition?

A

Antibodies, DNA and transcription factor, receptors, etc.

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3
Q

What is the general equation for ligand binding?

A

P + L –> PL

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4
Q

What type of interaction is ligand binding?

A

A noncovalent interaction

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5
Q

What is the formula for Ka (association constant)?

A

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

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6
Q

What is the formula for Kd (dissociation constant)?

A

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

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7
Q

What is the relationship between Kd and Ka?

A

Kd = 1/Ka

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8
Q

What are the units of Kd?

A

M

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9
Q

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

A

Strong association (weak disassociation)

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10
Q

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

A

Weak association (strong dissasociation)

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11
Q

In a cell, what does Kd reflect?

A

The concentrations in a cell

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12
Q

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

A

~ 1 nM (same order of magnitude)

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13
Q

What is the Kd of enzyme-ATP?

A

Millimolar to Micromolar

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14
Q

What is the Kd of signaling protein to a target?

A

Micromolar

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15
Q

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

A

Nanomolar

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16
Q

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

A

Nanomolar to picomolar

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17
Q

What is the Kd of biotin binding to aviding?

A

Femtomolar (one of the strongest noncovalent interactions)

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18
Q

What is specific binding limited by?

A

The number of available sites

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19
Q

What is a hallmark of specific binding interactions?

A

Saturable binding

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20
Q

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

A

Exponential saturation

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21
Q

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

A

Sigmoidal

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22
Q

How can Kd be determined from L concentration?

A

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

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23
Q

What is [L]?

A

Concentration of FREE ligand

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24
Q

What is [P]?

A

Concentration of FREE protein

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25
Q

What is [PL]?

A

Concentration of PL complex

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26
Q

What is [L]T?

A

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

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27
Q

What is [P]T?

A

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

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28
Q

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

A

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

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29
Q

What happens if [P]T < Kd?

A

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

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30
Q

What happens if [P]T > Kd?

A

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

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31
Q

What is f?

A

Fractional occupancy

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32
Q

What is the equation for f?

A

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

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33
Q

What happens at f = 0.5?

A

Kd = [L]

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34
Q

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

A

The [L] where f = 0.5

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35
Q

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

A

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

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36
Q

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

A

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

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37
Q

How can f be experimentally calculated?

A

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

38
Q

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

A

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

39
Q

What thermodynamic quantity changes upon ligand binding?

A

Delta G

40
Q

What is delta G bind?

A

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

41
Q

What is the formula for delta G bind?

A

Delta G bind = RT ln(Kd)

42
Q

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

A

It is more negative (higher magnitude)

43
Q

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

A

It is less negative (lower magnitude)

44
Q

What is the formula for delta G?

A

Delta G = delta H - T delta S

45
Q

What does delta H come from in ligand binding?

A

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

46
Q

How does ligand binding decrease entropy?

A

There is less degrees of freedom, hydrophobic effect

47
Q

How does ligand binding increase entropy?

A

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

48
Q

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

A

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

49
Q

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

A

S, because there are compensating effects

50
Q

How does the filter binding assay work?

A

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

51
Q

How is Kd determined from a filter binding assay?

A

Plot f and L to find Kd

52
Q

How does fluorescence polarization / anisotropy work?

A

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

53
Q

How is Kd determined from fluorescence polarization / anisotropy?

A

Can plot tumbling changes, and fit to a Kd

54
Q

How does FRET work?

A

Direct observation of two probes (see changes in concentration)

55
Q

How does the bead halo assay work?

A

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

56
Q

What are some methods to measure Kd?

A
  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)
57
Q

What is the general method for ligand binding assays?

A

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

58
Q

What concentration ranges are needed for a Kd assay?

A

A concentration range where Kd is in the middle

59
Q

What is the purpose of saturation measurements?

A

Bound fraction remains small

60
Q

Why is radioactivity used in ligand binding?

A

Only way to not change chemistry of ligand

61
Q

How is retinoic acid used in ligand binding?

A

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

62
Q

What is the purpose of radioactivity with retinoic acid?

A

Calculate radioactivity to measure free ligand

63
Q

What assumption is used in the retinoic acid test?

A

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

64
Q

What is retinoic acid?

A

Metabolite of vitamin A

65
Q

When is the Scatchard analysis done?

A

Estimate Kd when receptor concentration is unknown

66
Q

How does the Scratchard analysis work?

A

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

67
Q

What is the formula for Scatchard analysis?

A

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

68
Q

What is the slope of Scatchard analysis?

A

-1/Kd

69
Q

What is the x intercept of a Scatchard analysis?

A

[P]T / Kd

70
Q

What does the Hill equation describe?

A

The binding isotherm

71
Q

What is the Hill equation?

A

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

72
Q

What is n?

A

Hill coefficient

73
Q

What happens when n = 1?

A

No cooperativity

74
Q

What happens when n < 1?

A

Negative cooperativity

75
Q

What happens when n > 1?

A

Positive cooperativity

76
Q

Where does the biggest change of f occur?

A

Between 0.1 and 10 of L/Kd

77
Q

What is the chemical equation for competition?

A

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

78
Q

What does the Cheng-Prusoff equation describe?

A

Relate IC50% to Ki (more standardized value)

79
Q

What does ITC stand for?

A

Isothermal titration calorimetry

80
Q

What is ITC used for?

A

Measuring thermodynamics of binding?

81
Q

How does ITC work?

A

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

82
Q

How does ITC measure delta G?

A

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

83
Q

What is the formula for ITC Kd?

A

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

84
Q

How does ITC measure delta H?

A

It uses the heat released

85
Q

How doe ITC measure delta S?

A

It uses delta G and delta H to calculate delta S

86
Q

What is lock and key binding?

A

No change in conformation

87
Q

What is induced fit binding?

A

There is a conformational change in the protein

88
Q

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

A

Binding of a ligand can select one state over another

89
Q

What is ligand induced folding?

A

Binding of a ligand changes the protein shape

90
Q

What is conformational selection?

A

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

91
Q

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

A

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

92
Q

What is the Cheng Prusoff equation?

A

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

CHE 541 (20 decks)

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