Ligand Binding By NMR Flashcards
Explain the speeds across absorption , flurorense, NMR
Absorption fast (femtoseconds)
Fluorescence longer (nanoseconds)
NMR longest (milliseconds, lots of time for to do experiments)
What are the pros of NMR
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
Cons of NMR
Very expensive to maintain equipment
Highly specialized technique (not like DSF)
Explain how spins are measure in NMR
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
Explain the energy difference of the atoms in the magnetic S field
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)
Explain the delta E in nmr
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
What happens if the delta E is small in nmr
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)
How do you calculate the ratio of spin states in nmr
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
What does a 1D H NMR spectra of protien, DsDNA, polysaccharide show
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
Why would ser and thr have a diff chemical shift in their C beta hydrogen than the regular C beta hydrogen
They have an OH on the C beta
Why do we usually do nmr in D2O and not regular water
Because we can then remove the H spin signal from water since d2o spin isn’t measured
What is spin spin coupling and the impact of smaller molecules in nmr
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
Explain the machine setup in nmr and the axis
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
Explain the NMR radio frequency diagram at equilibrium
Excess spins
When 90 degree pulse added
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
Explain what happens if a 180 degree (or two 90 degree pulses) pulse is added in NMR
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
What do the actually measure in the nmr after the pulse is added
The return to equilibrium which inclides
- Spin lattice relaxation
- Spin spin relaxation
What is spin lattice relaxation
Whag is spin spin relaxation
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)
What does the relaxation of the spins lead to
What is this called
A decrease in the radio frequency signal/intensity
Free induction decay
What is free induction decay
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
What does doing a Fourier transform do in NMR
Turns the signal vs time plot of the free induction decay into the frequency domain to give us the actual 1D nmr spectrum
Why do we use isotopes in nmr
What is special about deuterium
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
Whag is special about the 1H in nmr
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
What is her reason for thinking nmr is a non sensitive technique
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
What elements are mostly used for nmr
1H, 2H, 13C, 15N, 19F, 31P
Don’t need isotope for the fluting because has high sensitivity
What does the protien sample have to be for NMR
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
Explain the multidimensional nmr
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
What is HSQC
Which do we use most
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)
Why do we use 2D nmr instead of 1D
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
Explain what a 2D 15N HSQC looks like
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
Where would we see more peaks in HSQC 2D spectrum , the 13C or 15N
The 13C because that are more carbons the nitrogen in protiens
What are assignments in HSQC
Assigning each of the peaks in the 15N- 1H 2D NMR to the specific amino acid they correspond to
What is great about nmr in terms of measuring binding
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
How can you examin a ligand binding NMR plot
The more caterpillar looking colourful dots show fast binding
The dots the don’t move much show slow binding
Come back to this
What are the three ways to do ligand binding experiments in NMR
Level 1: detect binding
Level 2: locate the binding site
Level 3: binding can cause large conformational changes
What does Level 1: detect binding mean in NMR ligand binding
See changes in chemical shifts and intensities of the peaks
Can’t figure out much more than the fact that it a ligand binds
What does Level 2: locate binding site mean in NMR ligand binding
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
What does Level 3: binding can cause large conf changes mean
If the ligand binds, conformation changes
This means we have to reassign the peaks from the protien because shift are diff now
What value of Kd indicates weak binding
Kd in mM range
Explain the theoretical NMR binding curve
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
Explain the chemical exchange peaks in NMR
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
When measuring ligand binding in NMR what is the NMR sensitive to
The off rate k
Explain the off rate and chemical shift difference relationship in 2D 15N- HSQC NMR ligand binding
Fast exhange: k»_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
What is kon controlled by
What does this do to the Kd equation
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
What is Kd when fast intermediate and slow exchange
Fast: Kd low, submicromolar (less than 1 micromolar)
Slow: Kd high, micromolar to mM
Intermediate: Kd micromolar
When they get the ligand binding N-HSQC spectra how do they actually get it
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
What do we plot of its a fast exchange
If slow exhange
The chemical shift
The intensity change (the peak disappears and reappears elesewhere)
Why would you reassign the spectrum during ligand binding
If there are singificant chemical shift changes from high affinity or slow exchange rates
Idk
How is NMR ligand binding done for larger protien complexes
Use isotope labeling to label one of either the protien or ligand and not the other
Then see the chemical shift upon binding
What is SAR by NMR
Structure activity relationships by NMR
Basically to makes drugs
Looks for high affinity ligands for protiens, then the ligands could be used in drugs
Explain the SAR by NMR process
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
In the ligand bindings with NMR what colour is the unbounds protien and the blind protien
Black unbound
Red bound
Whag does SAR do to the Kd
Gives ligand that have nanomolar Kd (smaller than micro, very tight binding)
How are sequence specific resonance assignments intially started
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
Explain the strips for the sequence specific resonance assignments
How do you analyze them
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
How can you tell from the strips for the sequence specific resonance assignments what alanine ser/thr and glycine look like
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
