Lec 29 Flashcards
What are the irreversible alkylating agents
Iodoacetate and iodoacetamide
What do Iodoacetate and iodoacetamide alkaylate at a slower rate
lys His Met
What makes Iodoacetate and iodoacetamide react faster and with what
How is this good
How do all the cys get alkylated
If the ph is higher than 6, the reaction with cys is faster
Can lead to specific alkylation
If add 8M urea and then reduce it at the same time
Why do Iodoacetate and iodoacetamide need to have DTT or BME removed before the reaction
Since DTT AND BME have sulfur groups, theyd react with the Iodoacetate and iodoacetamide
So DTT and BME need to be removed
How does Iodoacetate and iodoacetamide change the charge of a cys when alakylating it
Iodoacetate makes it go from neutral to negative
Iodoacetamide makes it go from neutral to neutral
What makes alkylation of residues good (how does the alkylation help)
When a crystal is formed, water is being excluded from the system thus increasing entropy
So to make a crystal more easily formed and formed faster, alkylation allows flexible amino acids like arg and lys to become more rigid
By doing this you do entropy reduction on the arg and lys
When more rigid, the proteins is better able to form a crystal for x ray crystallography
What are fab and why are they made
Cleaved antibodies which we can use to study the antibodies
How can iodoacetimide be used to make fab
What version do we use
Papain cys protease cleaves the antibody to make fab
The reaction gets stopped with iodoacetamide (we use the neutral version to prevent adding charges)
Why do we use iodoacetamide to stop the reaction of making fab
Because of kept going the papain will start cleaving other residues that we don’t want it to cleave
What is peptide mapping analysis and how is it done
You can identify what residue is being modified using mass spec
You have the native folded protein, It gets denatured, Cleave it with a protease through trypsin digest
Run the charged peptides through a mass spec
Do again but with the modified protien
Should see the loss of one peak and gain of a new peak to see which fragment was modified when comparing to regular protein spectrum
What is protein cross linking and why
What is used
We use it to see if two poly peptides or if two amino acids in a polypeptide are near each other
A bifunctional reagent: made of two reactive electrophillic groups joined by a linker
What are the two types of cross linking reagents and what do they mean
Homo bifunctional: one type of specificity at each end
Heterobifunctional : two types of specificity at each end (two types of functional groups)
What is the linker in a cross linking reagents
It’s either permanent or cleavable
If cleavable, you can cross linking two proteins to the cross linking reagent then cleave the linker that connects them
What can the length of the linker tell us within a poly peptides
The length of the linker determines the max distance between residues that get cross linked in the polypeptide
What is cross linking useful for within a polypeptide
What can this usefulness be applied to
What’s the downside
It can stabilize the tertiary structure by cross linking flexible regions to make the protein more rigid
Crystallography, cryo EM
Lose insight into the stability and structure of the protein since enforcing rigidity
What is cross linking between polypeptides useful for
- Protein protein interactions: can tell us about all the proteins surrounding the protein and what its interacting with
- Can help measure distance between polypeptides: usually use lys specific cross linkers since on the surface of proteins
- Can attach the protein to a carrier:
such as resin bead) to make a custom affinity chromatography column
Or for immunogenicity where you bind the protien that has low immune response to one that has a high immune response, then get a really good antibody response
What are three examples of cross linkers
Glutaraldehyde
DTSSP
APDP
What is glutaraldehyde
A homobifuntional cross linker
Reactive with lys and cis, random and not very specific cross linker
Can polymerize with itself to makes diff lengths of itself. So it has extensive cross linking. This is bad because can’t get reliable information about distances anymore
Volatile: good because if making crystal and add it to precipitant solution, it forms vapour and SLOWLY cross links molecules inside the crystal. Help crystal diffract better
What is DTSSP
Homobifunctional
lys specific crosslinker
Because it has a negative charge, it can’t enter membranes
Can cleave it with disulfide reducing agents (breaks the disulfide bond in the linker region)
Describe the structure of DTSSP and how it reacts with the lysine on a protein
Has a s-s in the linker region
Reactive portion at the ends get attacked by the NH2 on the lysine
Reactive portion leaves and protein with lysine is added
How can you use DTSSP to find protein complexes
React it in a mix of proteins (ABCD)
Proteins close to each other get cross linked
Use SDS page to see the change
With low DTSSP see all bands but also the heavy band
If both A and B bands disappear and one very high MW band is present, that means they got cross linked and are interacting
W
What it’s important to note for cross linking experiment
If ran too long, can lead to non specific binding beings protiens will move to get closer even when not interacting
This is why need to run controls and many runs of the experiment
What is APDP
A heterobifunctional linker
Non specific (binds to everything)
21 A long (very specific length to know distances between residues)
Has a pyridylthio group (cleavable linker reacts with cys or reducing agents to get cleaved)
Has photoreactive phenyl azide group: the azide (N3) reacts with the protien
The N3 is very reactive at 265 - 275 nm so it binds to many proteins
This cleavage makes a short lived phenyl nitrene group
Has an rafiolabelled 125 iodine on the phenyl ring
How can APDP be used to see protein protein interactions
Because the crosslinker is light sensitive, react it in the DARK with the protein of interest
Mix it with the other proteins
Treat with Uv light to cross link the 125 I to binding partners ex A AND B
Cleave off linker using reducing agent
Now the binding parent has the radio label iodine
Do SDS page and autoradiography (Only b shows up as a band due to radio label on it
So we know A was interacting with B
What is integrative structural biography
How do we do it
Way to get structural info of a protien using two or more structural methods
use one low resolution method to see interactions and combine it with a high resolution method
Ex. Cross linking (low res) with cryo EM (high res)
Negative staining with cryo em
What are high and res low res techniques
NMR, XRAY, CRYO EM
SUBTOMOGRAM, light microscopy, MICRO ED
How would you carry out a integrative structural biology analysis
Want to see the protein forming a complex
So you have the coarse grained representation (made high res individual protien components to low res blob of the protein)
This is to represent the size of the protein (speeds up the calculation because don’t have multiple parameters
And you combine this low res info to high resolution info (ex the cryo em map of the general shape of the protien complex)
To find how to best fit each high res proteins into the low res blob proteins (just trying diff combos to see which fits best in which blob)
Can help the computer with them better through cross linking experiment that says a protein component is interacting with another component
Gives many more specific models where some fit better than others
Computer give score on the models and you use score to predict structure
Why is integrative structural biology good
Good if you can’t get the high res structure using other methods
