Lec 26 Flashcards
What type of reaction is important for all the assumptions we make in the ligand binding equation
1:1 binding experiment
First order kinetics
What is KD when Y is 1/2
When Y=1/2
KD = [L]
So when
So on a plot if we have y=0.5 what is the KD
The x value at y=0.5 is KD which is also the [L]
What is the simplest definition of KD
KD= [L]
What has the highest affinity (lowest KD)
How can this be useful
Streptoavodin-biotin
Can tag something with biotin then use avodin beads to purify it
What is typically in the lower affinity range and why
Enzyme substrate binding
Because the substrate need to be released
What determines the strength of binding
The complementarity between the ligand and the binding site
What is included in complimentary
The size
Shape
Charge
Hydrophobicity
All of these come together to give a stronger interaction. (KD in the picomolar)
What are the models of binding
Lock and key
Induced fit
Conformational selection
What is the lock and key binding
Like a puzzle piece
What is the induced fit binding
The binding site changes it confirmation as it’s forming the complex
What is confomational selection
Give example
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
Is induced fit entropically favourable or disfaviurable
So what has to happen
Disfavourable
Has to be offset by a contribution by Enthalpy (like hydrogen bonds)
Units for KA/KD and KON/KOFF
KA M^-1
KD M
KON M-1SEC-1
KOFF sec-1
When does KD show us strong binding vs weak binding
<100nM strong
> 100 micromolar (weak)
What does delta g need to be for a reaction to occur
What does this mean a
Negative delta g
This means that either delta H needs to be very negative or delta s need to be very high
What is the equation for delta G
Delta G = delta H - T DELTA S standard
In a reaction can you have delta H near zero
What does this mean
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
What is delta G in relation to KD
Delta G standard = -RT ln (KD)
What experiments can measure apparent KD (approximate KD)
ELISA
FRET
MST
What are the pros and cons of ELISA
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.)
What are the pros and cons of FRET
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
What are the pros and cons of MST
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
How can KD be measured directly
SPR
ITC
What are the pros and cons of SPR
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
What are the pros and cons of ITC
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)
What do you really need to account for when measure the binding of ligands
The ligand and the protein need to be in the same buffer conditions (same ionic strength and ph)
How does SPR work
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
What actually changes the plasmons in SPR
The mass change of the ligand binding to analyte on the chip
Changes refractive index of the solution
Which changes the plasmons
In SPR what is the downside in terms of masses
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
What chip is used in SPR
Gold
EXPLAIN the SPR curve
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)
So in SPR how do you get KD
The Koff/Kon from the curve
What is SPR so useful for and why
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
If KD is uppercase what is it referring to
If lower case kd
If uppercase it refers to the concentration (M)
If lowercase refers to the rate (sec-1)
What are the issues with SPR 43:06
- 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
- Mass transport limitation: if the analyte is quickly on or off it’s hard to detect the binding
- Nonspecific binding: something that binds to the chip but isnt the ligand, leads to false positives and noise
- Don’t get stoichiometric info
- One to one binding model
What interferes with the signal in SPR
Membrane proteins
What does info does ITC give you
If delta H negative
If delta H postive
SGH AND KD
Losing heat
Absorbing heat
In ITC you what does the machine include
You have a reference cell and a sample cell
Then a sensor in between
How does the ITC machine work
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
How do you actually get delta H from the ITC curve
The area under the curve
On an ITC plot where do you find KD
In the middle of the curve
(Inflection point)
How do we get delta S in ITC
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
What is the temp in ITC
Isothermal so constant temp
