Ligand Binding By NMR

Question 1

Explain the speeds across absorption , flurorense, NMR

Answer 1

Absorption fast (femtoseconds)

Fluorescence longer (nanoseconds)

NMR longest (milliseconds, lots of time for to do experiments)

Question 2

What are the pros of NMR

Answer 2

Long lifetime of excited magnetization (long time before relaxation) (milliseconds)

Specific: only the nuclear and electron spins from the atoms absorb in the magnetic field

Has high abundance of protons that allow the method to work (due to the spins)

Sensitive to the chemical environment , diff chemical shift for the hydrogen depending on what is surrounding it

Low energy technique, meaning it’s non destructive

Versatile : can do multidimensional experiments

Question 3

Cons of NMR

Answer 3

Very expensive to maintain equipment

Highly specialized technique (not like DSF)

Question 4

Explain how spins are measure in NMR

Answer 4

The magnetic field is applied

The atoms direct themselves with the field

Pulse, go in opp direction

Remove pulse, spin back to magnetic field directions

We measure the spin back to the fields

Question 5

Explain the energy difference of the atoms in the magnetic S field

Answer 5

When magnetic field is zero the two spin states are at the same energy level

As moving to a higher magnetic field the energy difference between the two spin states scales linearly with the field (linear spin down and linear spin up)

Question 6

Explain the delta E in nmr

Answer 6

The energy differences between the two spin states are small which is why it’s corresponding to the radio frequency (MHz) part of the spectrum

Question 7

What happens if the delta E is small in nmr

Answer 7

That means that there is a similar number of nuclei in each of the two up or down energy states

So the ratio of nuclei in the up or down spin state (spin up and spin down) is closer to 1 (since similar amount in both)

Question 8

How do you calculate the ratio of spin states in nmr

Answer 8

Find delta E:
delta E = hv = ___ J

h in units of J/Hz and v in Hz

Find ratio:

Use equation , get decimal, put over 1,000,000

Question 9

What does a 1D H NMR spectra of protien, DsDNA, polysaccharide show

Answer 9

Chemical shift in ppm

Protien has complicated spectrum

DNA has less protons so less peaks than the protien which is why we usually measure protiens in NMR not dna

Polysaccharide (carbons with a lot of OH) has poor separation between peaks because the H all have the same chemical shift

Question 10

Why would ser and thr have a diff chemical shift in their C beta hydrogen than the regular C beta hydrogen

Answer 10

They have an OH on the C beta

Question 11

Why do we usually do nmr in D2O and not regular water

Answer 11

Because we can then remove the H spin signal from water since d2o spin isn’t measured

Question 12

What is spin spin coupling and the impact of smaller molecules in nmr

Answer 12

The nuclear spins of atoms interact magnetically Kd they are within a few binds of each other

This leads to spin spin coupling where you get multiplets on the plot

For smaller molecules it’s harder to distinguish the peaks in these multiplets because they overlap

Question 13

Explain the machine setup in nmr and the axis

Answer 13

Have the tube with sample in between two magnet poles

There is a transmitter (pulsing with this) and a receiver (picks up the signal) around the sample

X direction is up, Z depends on magnetic field, y is front

The z axis is aligned with the magnetic field (if fields side, z side)

The receiver detects radio frequency in the x y plane

Question 14

Explain the NMR radio frequency diagram at equilibrium

Excess spins

When 90 degree pulse added

Answer 14

The equivalent nuclei have their spins either align with the field or against the field

These all precess (rotate) at the same frequency not the same phase, meaning the arrows are in different directions

This is why there is nothing to detect in the x y plane since the diff random phases canceled out to be zero

The excess of spins with the field are left

When the pulse is added, excess spins are no longer in equilibrium, they shift against the magnetic field (x axis)

Now the phases are all aligned and there is something to detect in the x y plane

Then overtime they return to equilibrium with many different phases

Question 15

Explain what happens if a 180 degree (or two 90 degree pulses) pulse is added in NMR

Answer 15

The excess spins are fully inverted into the opp to magnetic field

There is no net magentization in the xy plane , so nothing to detect

Question 16

What do the actually measure in the nmr after the pulse is added

Answer 16

The return to equilibrium which inclides

1. Spin lattice relaxation
2. Spin spin relaxation

Question 17

What is spin lattice relaxation

Whag is spin spin relaxation

Answer 17

The exchange of energy between the nuclear spins and the surroundings (transferring energy to surroundings)

The exchange of energy between the nuclear spins and the neighbouring spins (coupling, transferring energy to the other spins to relax)

Question 18

What does the relaxation of the spins lead to

What is this called

Answer 18

A decrease in the radio frequency signal/intensity

Free induction decay

Question 19

What is free induction decay

Answer 19

Free: not dependent on the radio frequency

Induction: the magnetic signals induce the signal in the receiver (to detect the magnetic signal)

Decay: theres decrease in the signal overtime

Question 20

What does doing a Fourier transform do in NMR

Answer 20

Turns the signal vs time plot of the free induction decay into the frequency domain to give us the actual 1D nmr spectrum

Question 21

Why do we use isotopes in nmr

What is special about deuterium

Answer 21

For example c13 instead of c12

Because they have the spin of 1/2 that can be detected

Deuterium is 2H and has a spin of one so not measured in nmr

Question 22

Whag is special about the 1H in nmr

Answer 22

It’s the most abundant and so is used as an indicator of the relative sensitivity

We say the sensitivity of the machine to the hydrogen is 100 since it’s so abundant

Question 23

What is her reason for thinking nmr is a non sensitive technique

Answer 23

Because we need a high concentration of protien solution in the nmr tube usually 1mM

There is low natural abundance of the 15N and 13C, so we overproduce these in minimal media with nutrients enriched these isotopes (like 13C glucose)
can’t have anything else in the media so it only ales up that 13C

If you don’t do this, the concentration of the protien has to be higher to account for the low abundance , meaning if has to be soluble to get this >1mM concentration

Question 24

What elements are mostly used for nmr

Answer 24

1H, 2H, 13C, 15N, 19F, 31P

Don’t need isotope for the fluting because has high sensitivity

Question 25

What does the protien sample have to be for NMR

Answer 25

Homogenous (>90% pure)

Filtered: to remove particles, stop aggregation, to sterilize the sample (stop bacteria from growing in tube)

Also all additives like reducing agents , detergents (to solubilize membrane protiens), buffers need to be screened because the protien signal can change with these additions

Question 26

Explain the multidimensional nmr

Answer 26

Have the chemical shift for the protons on C and N in the x and y axis

The diagonal line shows peaks coming from individual 1D spectrums

If we want to see where a peak is coming from, split the diagram into four 1D plots

These plots are looking at diff chemical shifts for the carbon

For example we see a dot in the H-Cb chemical shift that means the peak in the 2D spectrum is coming from that

Then look at the chemical shift for the nitrogen, see peak in the H-Na

So the 2D spectrum dot is coming from the coupling between H-Cb and the H-Na

When coupling they relax at the same rate and give the same chemical shift which is why they make a single dot on the 2D nmr

Question 27

What is HSQC

Which do we use most

Answer 27

Heteronuclear single quantum coherence

Shows
13C -1H
15N - 1H

15N HSQC is used to test if a protiens is suitable for further experiments (look for a dispersion of signals, means good protien for further analysis)

Question 28

Why do we use 2D nmr instead of 1D

Answer 28

The 1D is very complicated and it’s hard to distinguish peaks

In 2D NMR you get more spread of peaks since they all don’t have the same chemical shift

This way you can assign the peaks

Question 29

Explain what a 2D 15N HSQC looks like

Answer 29

Have the 1H chemical shift on the x and 15N in the right

For the H shift , Only looking at the ppm chemical shift range of protons on
nitrogen’s, 5.5 to 10.5

The NH2 on asn and gln are at the top of the spectra and have two peaks at the same N ppm shift value but not the same H value

This is because those two hydrogens on the N are seeing the same nitrogen (so same nitrogen ppm) but still diff for the h chemical shift because the h are in diff positions

Question 30

Where would we see more peaks in HSQC 2D spectrum , the 13C or 15N

Answer 30

The 13C because that are more carbons the nitrogen in protiens

Question 31

What are assignments in HSQC

Answer 31

Assigning each of the peaks in the 15N- 1H 2D NMR to the specific amino acid they correspond to

Question 32

What is great about nmr in terms of measuring binding

Answer 32

NMR is able to detect weak binding (other techniques can’t detect weak binding)

Can find Kd

Can find the location and number of binding sites: because the chemical shift depends on the environment, when the ligand binds that affects the environment of the protien

For ligand binding Can use small sample sizes and low concentrations

Question 33

How can you examin a ligand binding NMR plot

Answer 33

The more caterpillar looking colourful dots show fast binding

The dots the don’t move much show slow binding

Come back to this

Question 34

What are the three ways to do ligand binding experiments in NMR

Answer 34

Level 1: detect binding

Level 2: locate the binding site

Level 3: binding can cause large conformational changes

Question 35

What does Level 1: detect binding mean in NMR ligand binding

Answer 35

See changes in chemical shifts and intensities of the peaks

Can’t figure out much more than the fact that it a ligand binds

Question 36

What does Level 2: locate binding site mean in NMR ligand binding

Answer 36

you have the peaks for 15N -HSQC assigned to know what amino acids are changing upon binding

Titrate with the ligand

the changes that are seen are due to the ligand binding but the ligand isnt labeled so it shows no peaks in the spectrum

These changes in the environment upon binding change the spin, change the chemical shifts and/or intensity

The changes depend on the off rate of the ligand and the chemical shift difference between free and ligand bound states

Question 37

What does Level 3: binding can cause large conf changes mean

Answer 37

If the ligand binds, conformation changes

This means we have to reassign the peaks from the protien because shift are diff now

Question 38

What value of Kd indicates weak binding

Answer 38

Kd in mM range

Question 39

Explain the theoretical NMR binding curve

Answer 39

Chemical shift diff vs ligand concentration

At lower Kd (micromolar), tighter binding of ligand, the chemical shift diff is higher

At higher Kd (mM) less binding to ligand, the chemical shift diff is lower

Question 40

Explain the chemical exchange peaks in NMR

Answer 40

If it’s a slow exchange the P peak and the PL peak have a higher chemical shift diff than the off rate , so they are more separated peaks

If it’s a fast exchange the P peak and the PL peak have lower chemical shift diff than the off rate, and look like one peak

If intermediate exhange the P peak and the PL peak broaden and start to show two broad peaks or one broad peak

Question 41

When measuring ligand binding in NMR what is the NMR sensitive to

Answer 41

The off rate k

Question 42

Explain the off rate and chemical shift difference relationship in 2D 15N- HSQC NMR ligand binding

Answer 42

Fast exhange: k&raquo_space;> delta F, the peaks shift positions like a blur graudually (so each titration of ligand shift the circle over a bit)

Slow exhange : k «< delta F, the peaks is in one place then reappears in another spot

Intermediate exchange: k ~~ delta F, peak broadens and shifts, could also vanish

Question 43

What is kon controlled by

What does this do to the Kd equation

Answer 43

Diffusion controlled, so usually 10^9 M-1s-1

As fast as the ligand can get to the protien it binds

Kd = koff / kon

Kd = koff/ 10^9

So koff= 10^9 Kd

Question 44

What is Kd when fast intermediate and slow exchange

Answer 44

Fast: Kd low, submicromolar (less than 1 micromolar)

Slow: Kd high, micromolar to mM

Intermediate: Kd micromolar

Question 45

When they get the ligand binding N-HSQC spectra how do they actually get it

Answer 45

Do titrations with the ligand

Each dot on the spectra shows a single titration and single N-HSQC corresponding to that titration

If saw 8 colours on the ligand binding spectra that is 8 diff titrations and spectra

Question 46

What do we plot of its a fast exchange

If slow exhange

Answer 46

The chemical shift

The intensity change (the peak disappears and reappears elesewhere)

Question 47

Why would you reassign the spectrum during ligand binding

Answer 47

If there are singificant chemical shift changes from high affinity or slow exchange rates

Idk

Question 48

How is NMR ligand binding done for larger protien complexes

Answer 48

Use isotope labeling to label one of either the protien or ligand and not the other

Then see the chemical shift upon binding

Question 49

What is SAR by NMR

Answer 49

Structure activity relationships by NMR

Basically to makes drugs

Looks for high affinity ligands for protiens, then the ligands could be used in drugs

Question 50

Explain the SAR by NMR process

Answer 50

Have protien with two binding sites

A low affinity ligand binds of a binding site (not perfect binding)

Then we optimize the ligand to have better binding (better fit)

To the same for the second ligand

Link the two ligands together on the protien to make them both higher affinity

Question 51

In the ligand bindings with NMR what colour is the unbounds protien and the blind protien

Answer 51

Black unbound

Red bound

Question 52

Whag does SAR do to the Kd

Answer 52

Gives ligand that have nanomolar Kd (smaller than micro, very tight binding)

Question 53

How are sequence specific resonance assignments intially started

Answer 53

Have the 2D HSQC nmr with 15N and 1H

Add in the value of the 13C

So H-N-C=O now

This makes a 3D spectrum where the dots now shift up to match the chemical shift value of the 13C

Shift up by diff amounts because diff 13C chemical shift for each dot

Now goes from 2D HSQC to 3D HNCO

Now in the 3D box, we take 2D slices and look at each chemical shift of the each dot/peak alone

This 2D slice is separated into strips with each dot inside

Question 54

Explain the strips for the sequence specific resonance assignments

How do you analyze them

Answer 54

Have the H on the front N on side and 13C on top of box

So each strip gives the ppm:
full range for 13C
a narrow range for 1H
single value for 15N

You lay all of the individual strips down to align each chemical shift ppm dot/peak on one strip with the previous one

Ex. Ppm at 9, strip next to it should have ppm at 9

Basically matching carbons that have the same chemical shift

The tol half of the strip of the c beta and the bottom half is the c alpha

The size of the dots in the strips corresponds to the size of the peak, bigger dot bigger peak, more coupling

Dots Going from big to small to big to small in each strip

The big dot to the small dot shows the high C alpha coupling, the smaller dot shows the weaker coupling of the c alpha to the NH

Same c alpha so same chemical shift, just diff intensity bc showing weaker coupling

Question 55

How can you tell from the strips for the sequence specific resonance assignments what alanine ser/thr and glycine look like

Answer 55

For ser/thr there would be no chemical shift peaks in the C beta region (top part) because ser/thr are a special case where their c beta shows up in the calpha region

So see two dots , c alpha c beta, in the c alpha region

IDK SHE SAID DOESNT MATTER