ITC and DSF

Question 1

What does ITC do

Answer 1

Uses titration of the ligand injected into the protien sample (ligand added to protien)

Measures the Enthalpy of binding of the ligand with the protien

To keep at a constant temp electrical energy from a heater is used

Slow experiment

Question 2

Why does we add ligand to the protien for KTC

Answer 2

Because it’s being titrating in you can use a higher concentration of ligand

Basically the stock solution

Question 3

What do we measure for ITC

What happens if delta H is negative

Answer 3

The qp (heat) = delta H of binding

If negative, less electrical energy is needed after the injections because heat is already being released

Question 4

In ITC what are we trying to make sure

Answer 4

That after each addition of ligand to protien the solution is at equilibrium and you leave ample time for this to happen before the next injection

Meaning in soliton there is free ligand, free protien, and LP

Question 5

Explain the results of ITC

Answer 5

Once injection, peak goes negative, then reaches equilibrium

Then next injection, reaches equilibrium

In the first four injections, all ligands bind giving the same amount of PL complex in each case, meaning the delta H is also the same

This is because there’s so much protien in there that all ligand binds to it

Then eventually there is partial binding so smaller peaks

Then no binding because all the protien binding sites are saturated with ligand

Question 6

Why do we still see small peaks in ITC when there is no binding happening

Answer 6

Still adding one solution to the other so there are heats of dilution that show up

Question 7

What do you have to make sure after each injection in ITC

Answer 7

make sure do mix to get the ligand and the complex evenly distributed

Question 8

What data do we collect for ITC

What assumption is made

Answer 8

The q (heat) for each injection (usually negative)

The q gets smaller after more injections (because less binding)

Since all ligand binds to protien in the first injection, assume that the mol of L•P = mol of L added (since the mol of L added is all the PL complex)

Question 9

What are the things to consider in ITC experiments

Answer 9

The sample has to be degassed to remove bubbles because they interfere with scans

Usually do a blank experiment first then the actual (not at the same time)

Have to consider the heats that come from dilution and solvation

Need to have enough time between injections to reach equilibrium before the next injection

Need a clean instrument so only the ligand and not contaminants are changing the enthalpy

Question 10

ITC Adavantages

Answer 10

Non destructive, label free

Finds delta H directly without assumptions

Gives changes in Cp (heat capacity) and gives Ka

Question 11

ITC disadvantages

Answer 11

Needs high concentration of L and P because we’re measuring small delta H (need to be able to see the small delta H)

The signals for non covalent complexes are weaker than covalent (so they release less H)

Overlapping peaks

Time consuming

If the delta H is zero you can’t measure anything (so your things need to have a delta H)

Question 12

What does DSF measure

Answer 12

The fluorescence from a fluorophore that increases when it’s bound to a hydrophobic region of a protien

Question 13

What does DSF characteristics

Answer 13

It’s thermal shift assay (looking at a change in temp)

Can show ligand binding (since the binding can change the temp)

It’s label free (non covalently bound fluorophore)

The instrument is common

Low expertise

High throughput (many sample can be run at once)

Question 14

What fluorophore do we use in DSF

Answer 14

SYPRO orange dye which we buy as a stock solution diluted 5000x in DMSO

The DMSO is has hydrophobic and hydrophilic regions so it can solublized things that wouldn’t normally be soluble

Question 15

Does SYPRO orange dye fluoresce when it’s not with the protien

Answer 15

No , need to bind to protien to have fluorescence

Question 16

Explain qPCR

Answer 16

A thermal cycler with a fluorimeter

Machine heats samples quickly for cycles of replication

Then illuminates the sample and detects the fluorencnwse after each of the cycles

Question 17

Explain qPCR

Answer 17

A thermal cycler with a fluorimeter

Machine heats samples quickly for cycles of replication

Then illuminates the sample and detects the fluoresces from a nonspecific intercalating dye or a sequence specific DNA probe after each of the cycles

Question 18

What does the qPCR detect

Answer 18

Double stranded DNA

Question 19

Explain the sequence specific DNA probe in qPCR for DSF

Answer 19

The probe has a Florensence reporter and quencher

It anneals to the ssdna like a primer and the Taq polymerase from PCR degrades the prove

This degredation causes separation of the quencher from the reporter causing fluorescence to increase

Question 20

Explain the DSF peak

Answer 20

Florence vs temp

Intact protien , no flour

As temp increases the intact protien melts and exposes its hydrophobic residues

The SYPRO dye can now start to bind , line going up on the graph shows melting temp

Peak happens when dye fully bound

Starts to go down when temp increases past because protein aggregates and the dye dissociates

Question 21

What can DSF be used for

Answer 21

To measure melting point

Find conditions that stabilize the protien:
we have protien in diff buffers (diff pH and molecules giving that ph) and ionic strength and see how the melting point changes

Can see if additives destabilize the protien: if want to measure ligand binding you need the ligand in DMSO, see if DMSO changes something

Protien engineering: seeing what enzymes to add to detergents, want that enzyme to be stable at higher temp

Ligand binding: if ligand binds, increase the Tm since increase stability

Question 22

What are the disadvantages for DSF

Answer 22

Can’t get delta H, delta S, or stoich

Only get the melting temp