Lec 29

Question 1

What are the irreversible alkylating agents

Answer 1

Iodoacetate and iodoacetamide

Question 2

What do Iodoacetate and iodoacetamide alkaylate at a slower rate

Answer 2

lys His Met

Question 3

What makes Iodoacetate and iodoacetamide react faster and with what

How is this good

How do all the cys get alkylated

Answer 3

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

Question 4

Why do Iodoacetate and iodoacetamide need to have DTT or BME removed before the reaction

Answer 4

Since DTT AND BME have sulfur groups, theyd react with the Iodoacetate and iodoacetamide

So DTT and BME need to be removed

Question 5

How does Iodoacetate and iodoacetamide change the charge of a cys when alakylating it

Answer 5

Iodoacetate makes it go from neutral to negative

Iodoacetamide makes it go from neutral to neutral

Question 6

What makes alkylation of residues good (how does the alkylation help)

Answer 6

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

Question 7

What are fab and why are they made

Answer 7

Cleaved antibodies which we can use to study the antibodies

Question 8

How can iodoacetimide be used to make fab

What version do we use

Answer 8

Papain cys protease cleaves the antibody to make fab

The reaction gets stopped with iodoacetamide (we use the neutral version to prevent adding charges)

Question 9

Why do we use iodoacetamide to stop the reaction of making fab

Answer 9

Because of kept going the papain will start cleaving other residues that we don’t want it to cleave

Question 10

What is peptide mapping analysis and how is it done

Answer 10

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

Question 11

What is protein cross linking and why

What is used

Answer 11

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

Question 12

What are the two types of cross linking reagents and what do they mean

Answer 12

Homo bifunctional: one type of specificity at each end

Heterobifunctional : two types of specificity at each end (two types of functional groups)

Question 13

What is the linker in a cross linking reagents

Answer 13

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

Question 14

What can the length of the linker tell us within a poly peptides

Answer 14

The length of the linker determines the max distance between residues that get cross linked in the polypeptide

Question 15

What is cross linking useful for within a polypeptide

What can this usefulness be applied to

What’s the downside

Answer 15

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

Question 16

What is cross linking between polypeptides useful for

Answer 16

1. Protein protein interactions: can tell us about all the proteins surrounding the protein and what its interacting with
2. Can help measure distance between polypeptides: usually use lys specific cross linkers since on the surface of proteins
3. 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

Question 17

What are three examples of cross linkers

Answer 17

Glutaraldehyde

DTSSP

APDP

Question 18

What is glutaraldehyde

Answer 18

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

Question 19

What is DTSSP

Answer 19

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)

Question 20

Describe the structure of DTSSP and how it reacts with the lysine on a protein

Answer 20

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

Question 21

How can you use DTSSP to find protein complexes

Answer 21

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

Question 22

What it’s important to note for cross linking experiment

Answer 22

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

Question 23

What is APDP

Answer 23

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

Question 24

How can APDP be used to see protein protein interactions

Answer 24

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

Question 25

What is integrative structural biography

How do we do it

Answer 25

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

Question 26

What are high and res low res techniques

Answer 26

NMR, XRAY, CRYO EM

SUBTOMOGRAM, light microscopy, MICRO ED

Question 27

How would you carry out a integrative structural biology analysis

Answer 27

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

Question 28

Why is integrative structural biology good

Answer 28

Good if you can’t get the high res structure using other methods