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Bcem 431 > Lec 26 > Flashcards

Lec 26 Flashcards

1
Q

What type of reaction is important for all the assumptions we make in the ligand binding equation

A

1:1 binding experiment

First order kinetics

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2
Q

What is KD when Y is 1/2

A

When Y=1/2

KD = [L]

So when

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3
Q

So on a plot if we have y=0.5 what is the KD

A

The x value at y=0.5 is KD which is also the [L]

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4
Q

What is the simplest definition of KD

A

KD= [L]

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5
Q

What has the highest affinity (lowest KD)

How can this be useful

A

Streptoavodin-biotin

Can tag something with biotin then use avodin beads to purify it

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6
Q

What is typically in the lower affinity range and why

A

Enzyme substrate binding

Because the substrate need to be released

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7
Q

What determines the strength of binding

A

The complementarity between the ligand and the binding site

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8
Q

What is included in complimentary

A

The size

Shape

Charge

Hydrophobicity

All of these come together to give a stronger interaction. (KD in the picomolar)

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9
Q

What are the models of binding

A

Lock and key

Induced fit

Conformational selection

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10
Q

What is the lock and key binding

A

Like a puzzle piece

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11
Q

What is the induced fit binding

A

The binding site changes it confirmation as it’s forming the complex

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12
Q

What is confomational selection

Give example

A

The enzyme selects the conformation of the Ligand or vice versa

Ex. A enzyme can exist in three diff states, the ligand selects a specific state

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13
Q

Is induced fit entropically favourable or disfaviurable

So what has to happen

A

Disfavourable

Has to be offset by a contribution by Enthalpy (like hydrogen bonds)

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14
Q

Units for KA/KD and KON/KOFF

A

KA M^-1

KD M

KON M-1SEC-1

KOFF sec-1

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15
Q

When does KD show us strong binding vs weak binding

A

<100nM strong

> 100 micromolar (weak)

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16
Q

What does delta g need to be for a reaction to occur

What does this mean a

A

Negative delta g

This means that either delta H needs to be very negative or delta s need to be very high

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17
Q

What is the equation for delta G

A

Delta G = delta H - T DELTA S standard

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18
Q

In a reaction can you have delta H near zero

What does this mean

A

Yes if the entropy is high enough to give the negative delta g

This is why we don’t need to rely on delta H

19
Q

What is delta G in relation to KD

A

Delta G standard = -RT ln (KD)

20
Q

What experiments can measure apparent KD (approximate KD)

A

ELISA

FRET

MST

21
Q

What are the pros and cons of ELISA

A

Pros: sensitive,
cheap,
quantitative,
can be adapted for high throughput screening (can do many ELISA at the same time)
Can be use to detect interactions in complex mixtures

Cons: its an indirect measurement that relies on enzyme conjugated antibodies (expensive)

Can lead to non specific binding which causes false positives (get a result that’s not true)

Doesn’t give kinetic data (like entropy Enthalpy etc.)

22
Q

What are the pros and cons of FRET

A

Pros: can observe protein interactions in live cells, which can help understand interactions in their natural environment

Cons: needs the labelling of the protein with the fluorescent tags which is technically challenging and affects the protein function

23
Q

What are the pros and cons of MST

A

Pros: it measures the motion of molecules in a temperature gradient, so the temp changes when there is binding, which give an idea of the affinity

This means it’s a label free method, needs small sample volumes

Cons: pricey, sensitive to changes in solution ionic strength or ph, this affect the movement of the molecules in the gradient

24
Q

How can KD be measured directly

A

SPR

ITC

25
Q

What are the pros and cons of SPR

A

Pros: Label free technique , give real time monitoring of the binding

Only technique that gives Kon and Koff and the Keq

Needs low sample prep, can use lower concentrations

Cons:
Expensive

Requires the sample to be immobilized on a chip, which changes its native conformation

Data analysis is complex

26
Q

What are the pros and cons of ITC

A

Pros:

Only measure heat change (delta H) during binding

Gives a direct measurement of KD and gives all of the H S AND G values without needing to label

Cons:

Need a large amount of sample

Not sensitive enough to measure weak interactions (since measuring delta h, if delta h is low, method suffers)

27
Q

What do you really need to account for when measure the binding of ligands

A

The ligand and the protein need to be in the same buffer conditions (same ionic strength and ph)

28
Q

How does SPR work

A

Immobilize a ligand on a chip (the conjugated ligand)

Before experiment, a laser shines onto the matrix with just the buffer and gives a refractive index based on the plasmons

The analyte is flowed into the chip (the enzyme) and binds to the ligand

A laser shines behind the matrix (chip) and generates plasmons at different angles

So The reflective index changes from just buffer to ligand with analyte bound

When the ligand binds, signal changes from buffer baseline to a different signal

Then when it comes off it goes back to the buffer baseline

this changing refractive index signal tells us Kon and Koff

29
Q

What actually changes the plasmons in SPR

A

The mass change of the ligand binding to analyte on the chip

Changes refractive index of the solution

Which changes the plasmons

30
Q

In SPR what is the downside in terms of masses

A

If you have a large difference in masses between the ligand and the analytes, you can’t measure the signal well

Need roughly the same masses between the two

31
Q

What chip is used in SPR

A

Gold

32
Q

EXPLAIN the SPR curve

A

Intially just the baseline buffer signal

When analyte binds, the response unit goes up (gives Kon)

Then reach equilibrium (which gives Keq)

Then comes off so response into starts to go down (Koff)

33
Q

So in SPR how do you get KD

A

The Koff/Kon from the curve

34
Q

What is SPR so useful for and why

A

In pharmacology drug binding

You want to know how long the ligand binds to the protien

This is given by Koff

SPR is the only method that gives Koff

35
Q

If KD is uppercase what is it referring to

If lower case kd

A

If uppercase it refers to the concentration (M)

If lowercase refers to the rate (sec-1)

36
Q

What are the issues with SPR 43:06

A
  1. Limited detection range: SPR is most sensitive to changes in the refractive index near the metal surface. So analytes with low MW or refractive index are hard to detect

If low change in the mass not good signal

  1. Mass transport limitation: if the analyte is quickly on or off it’s hard to detect the binding
  2. Nonspecific binding: something that binds to the chip but isnt the ligand, leads to false positives and noise
  3. Don’t get stoichiometric info
  4. One to one binding model
37
Q

What interferes with the signal in SPR

A

Membrane proteins

38
Q

What does info does ITC give you

If delta H negative

If delta H postive

A

SGH AND KD

Losing heat

Absorbing heat

39
Q

In ITC you what does the machine include

A

You have a reference cell and a sample cell

Then a sensor in between

40
Q

How does the ITC machine work

A

The titrant solution (the ligand) comes into the sample cell

Inside the sample cell is the analyte

Then you measure heat per unit time (power)

As binding happens there is heat released or absorbed

41
Q

How do you actually get delta H from the ITC curve

A

The area under the curve

42
Q

On an ITC plot where do you find KD

A

In the middle of the curve

(Inflection point)

43
Q

How do we get delta S in ITC

A

Indirectly, we don’t measure the entropy

From the curve you get KD (use delta g with KD equation to get delta G)

Then find entropy using the delta g and the delta H

Using delta H to find everything

44
Q

What is the temp in ITC

A

Isothermal so constant temp

Bcem 431 (26 decks)

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