Lec 28 Flashcards
In a water into water ITC isotherm why do we see heats
What does this show
It’s the same thing being one red into itself so shouldn’t see Change in heat
But when injecting something there is mechanical force so dispersion forces occur, causing new h bonds to be made, Chang in heat
It shows how the machine is very sensitive to small changes in heat which is why the buffer and the pH need to be exactly right
How can you tell based on an ITC plot if a reaction is endo or exothermic
If the peaks are going down
Releasing energy
Exothermic
What is the equation in ITC that describes the shape of the curve
What value doesn’t give accurate measurement of inflection points
What’s the best range
C= n x Ka x [cell]
Or C= n x [Cell]/Kd
n is the stochiometry of the binding usually assume 1
Cell is concentration of whatever your putting into the experiment
If C less than 5, the line on the plot would be shallow and can’t tell where inflection point is so all Kd, H and n stochiomety inaccurate
Also if too high >500, the S shape curve slope jumps to fast and can’t find Kd accurately, but only the Kd inaccurate
5-500
What is the purpose of doing a buffer control experiment along with the actual experiment
What else does this show
The control shows if the signal is changing even without the ligand due to buffer or salt mismatches
Comparing this to the actual , if you see any difference between the background and the experiment you can tell there there is binding without even needing a proper curve
This shows that if you have a vague idea of the affinity , you can find the see value to predict what the curve will look like
Why do we chemically modify protiens
To get structural of functional info of the enzyme
To find the most reactive group in the amino acid (usually ser thr cys most reactive, so these would be the first ones that are modified)
Can modified residues to see which are surface exposed
Can add radioactive tags or introduce spectroscopic probes
Can show protien interactions. Through cross linking
What are the experimental considerations when doing chemical modifications
The extent of the modifications (how long doing reaction)
The specificity and side reactions
The changes in conformation
What is meant by extent of modification
The amount of reagent you use, if add too much could be bad, or may need a lot to actually get it modified
The pH, reactivity of the side chain depends on its ionization state, this can be used to find pKa (ex low pH stops HDX)
Presence of denaturants, some modifications only work if the protien is denatured
What is meant by specificity and side reactions
Most modifying reagents react with more than one type of residue on the protien, want to choose the best conditions to stop this
Can do genetic engineering to mutate out or in residues, (ex if no cys mutate an amino acid to cys to get modification)
The lack of specificity might be good if you want to test the role of more than one type of residue
The active site could be very reactive leading to specificity
What is meant by changes in confirmations
When the protien is modified, it might denature because of the modification or the conditions of the chemical reaction, don’t want that
So you need to see if the change in conformation caused a change in the activity, if it does then the modification isn’t useful for info since there is random change in the activity not specific due to the modification
Reactive groups are
The modification reagents are
Nucleophiles like CMK HYE RW
Electrophillic
What are the reducing agents
BME
DTT
TCEP
What is BME
if more cys increase concentration
Don’t want to use too much
Volatile stinks
Half life 100 hours at pH 6.5, 4 hours at pH 8.5, so need to use fast
Toxic, so make sure it’s sealed
Covalent irreversible reaction
Explain the structure of BME and the reaction
What is special about the reaction
Has a SH and OH on either end, neutral
It covalently attached to one S of the disulfide bond
Then colelentlg attaches to itself a to make its own disulfide bond
Add acid to prevent reoxidation
In the middle step it could covlenetly modifiy the cysteine if doesn’t form the disulphides bond with itself
What are the two main ways to reduce something
The reducing agent can just break it
Or it can covelently attach to break it (meaning it could modifiy it as well by Covalently attaching)
What is DTT structure
Two sh and two oh, neutral
Less volatile than BME , stinks
Half life is 40 hours at 6.5 pH and 1.5 hours at 8.5 (so shorter half life)
Toxic, Stronger than BME
