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Proteins > Ligand Binding > Flashcards

Ligand Binding Flashcards

1
Q

What are the qualititative methods of observing ligand binding?

A 
Direct visualisation
Genetic complementation/yeast 2 hybrid TF
Fluorescence microscopy
FRET
Co-immunoprecipitation
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2
Q

What are the quantitative methods of observing ligand binding?

A

Biophysical radiolabel binding assays
Isothermal Calorimetry
Competition assays

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3
Q

What resolution can fluorescence light microscopy detect?

A

200nm

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4
Q

What distance can FRET detect?

A

2-8nm

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5
Q

What resolution is PALM/STORM microscopy?

A

20-100nm

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6
Q

Where is fluorescence microscopy used?

A

Light in living cells

PALM/STORM in fixed cells

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7
Q

Can fluorescence microscopy detect proteins?

A

No, resolution larger than 2-20nm proteins

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8
Q

Why is quantitative characteristation needed?

A 
Eliminate non-specific binding
Identify type of interaction
Determine affinity of interaction
Identify stability of complex
Understand regulation by PTM and effects of solution
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9
Q

What is the main problem with qualitative methods?

A

Don’t determine direct/indirect interaction

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10
Q

Which test is the qualitative “Gold Standard”?

A

Co-immunoprecipitation

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11
Q

How can co-immunoprecipitation be made quantitative?

A

PKA subunits can be separated by cAMP and bound using differently tagged fluorescent antibodies

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12
Q

What type of kinetics must be used for pull-down assays?

A

Slow reaching of equilibrium/Kd

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13
Q

What type of affinity has a -∆G?

A

Small Kd/ high affinity

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14
Q

Outline biophysical assay process?

A

Label ligand and characterise radioactive signal
Incubate with cells
Wash to remove NSB
Separate bound ligand from unbound
Cell and label counting
Determine control for NSB using excess ligand

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15
Q

How can a biophysical assay be used to find Kd?

A

Plot [Bound ligand] against [bound/unbound]

slope is -1/Kd

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16
Q

What factors limit a biophysical assay?

A

Complex is unstable for washing
Difficult to separate bound/unbound
NSB masking specific binding
Low receptor concentrations

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17
Q

How can low receptor concentrations be assayed?

A

Sub-stoichiometric approximation using rearranged equations

18
Q

How can a high affinity ligand be assayed?

A

Using a competition assay to observe [RL] as a function of [C]
Assumes [L=L0] and [C=C0],
Low [R] and low [L]
Kdapp ~ Kc

19
Q

What range of Kd can be detected in a competion assay?

A

1pM-1nM

20
Q

What is a label-free binding assay also called?

A

Isothermal calorimetry

21
Q

What range of Kd can be detected in a binding assay?

A

1nM-1mM

22
Q

How does ITC measure Kd?

A

Measures heat for ∆H
∆G= RTln(Kass)
Plot time against heat. Gradient is Kass

23
Q

What must be kept constant during ITC?

A

State variables: T, P, V, [C]

24
Q

What is the concentration dependance of ITC?

A

C=Ka[R]

25
Q

How can the concentration dependance of ITC be reduced?

A

Using weak inhibitors/competition

26
Q

What sensitivity is ITC set to?

A

Lowest [L] usable above “noise”.

Measures µJ scale

27
Q

What type of binding is spontaneous?

A

-∆G, -∆H, -∆S, Small Kd

28
Q

How does HIV protease inhibitor evade neutralisation?

A

Binds CD4 receptors tightly to order gp120 region

Conformational masking does not bind antibody

29
Q

What is the relationship between ∆G and Kd?

A

Large changes in Kd caused by small changes in ∆G

30
Q

What rate order are ligand binding assays?

A

Pseudo-first order

31
Q

How can equilibrium thermodynamics be used to determine rate?

A

rate of association and rate of dissociation for different [L].
Plot [L] against peak response
plot scatchard graph to find Kd

32
Q

What does surface plasmon resonance measure?

A

Measures binding kinetics

33
Q

How does SPR measure binding kinetics?

A

biosensor (gold) with immobilised ligand reflects polarised light at a different angle when in complex

34
Q

What is reaction rate controlled by?

A

energy barriers and collision rates

35
Q

What is the limit of reaction rate in barrier-less solutions?

A

10^9/M/s

36
Q

How can Kd be defined in terms of rates?

A

Kd = rate diss/ rate ass

37
Q

What effect does mass transport have on the kinetics?

A

Produces a rate slower then true

38
Q

What is bulk transport in SPR?

A

The difference between eqm RU and reference values

39
Q

What is 1RU in SPR?

A

1pg/mm^2 protein density

40
Q

How is SPR used with antibodies?

A

Can calculate affinity of polyclonal antibodies by competition

41
Q

How was SPR used to characterise HIV recognition by antibodies?

A

Split personalities have 2 different variable chains

Polyreactive/cross reactive bind with low affinity but low specificty

Proteins (12 decks)

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