NMR Flashcards
What are the pros and cons of biomolecular NMR
What information can be obtained from an NMR spectra
What is equilibrium magnetisation
How does 1D and 2D NMR work
Explain free induction decay (FID)
When a sample is exposed to a strong magnetic field, these nuclear spins align with the magnetic field in one of two possible directions (parallel or anti-parallel).
By applying a radiofrequency (RF) pulse, we flip the spins out of alignment with the magnetic field. This is a short, intense pulse of energy that excites the nuclei from their lower energy state (aligned) to a higher energy state (anti-aligned).
After the RF pulse is turned off, the excited nuclei relax back to their original alignment with the magnetic field. This relaxation process causes the nuclei to emit signals (radiofrequency signals) as they return to equilibrium.
The emitted signal is called the Free Induction Decay (FID).
The FID is a decaying oscillation because the nuclei do not relax instantaneously. The signal gradually diminishes over time as the energy is released.
Explain 1D and 2D homonuclear NMR
In 2D heteronuclear NMR, the same general scheme is used. We comment here the case of 1H-15N HSQC. After an initial preparation time, a 90° hard pulse in the 1H channel excites the 1H atoms (in NMR jargon referred as “protons”). The 1H excitation is followed by an INEPT element that exploits simoultaneous pulses between 1H and 15N channels to enable the transfer of the excitation (“polarisation”) from the 1H to the 15N. NOTE1: The reason why we excite the 1H first is that this nucleus is more sensitive because of the highest gyromagnetic ratio. NOTE2: It is beyond the scope of this course to know how and INEPT scheme is made. After the polarisation is transferred on the 15N, an evolution time t1 (indirect evolution) follows (T1 increases gradually during the experiment). After t1, the polarisation is transferred back to 1H via another INEPT. Detection is made during t2 (direct time). NOTE3: during acquisition on the 1H channel, the 15N is hit by a broadband RF to decouple 1H and 15N, otherwise we have peaks split in two.
Explain HSQC NMR*
Do 90 degree pulse
Then do INEPT - transfers magnetisation from proton to nitrogen
Then do T1 period with incremental delays
Then do another INEPT - transfers magnetisation from nitrogen to proton + detect on the proton
used to study heteronuclear interactions, particularly the correlations between protons (¹H) and heteronuclei (most commonly carbon-13 (¹³C) and nitrogen-15 (¹⁵N))
goal of HSQC is to identify which hydrogen atoms (protons) are directly bonded to a heteronucleus (e.g., carbon or nitrogen)
In an HSQC experiment, two NMR frequencies are measured:
The proton (¹H) frequency: This gives information about the protons in the molecule.
The heteronucleus (¹³C or ¹⁵N) frequency: This gives information about the carbon or nitrogen atoms directly bonded to the protons.
What nuclei can be used in heteronuclear NMR
Explain 1H-15N HSQC heteronuclear NMR*
Proton-Nitrogen Correlation: The technique directly correlates hydrogens attached to nitrogens (often in amide groups of proteins) in a 2D spectrum.
The resulting 2D spectrum will have:
The x-axis corresponding to the ¹H chemical shifts (protons).
The y-axis corresponding to the ¹⁵N chemical shifts (nitrogens).
Each cross-peak in the 2D spectrum corresponds to a pair of protons and their directly bonded nitrogen atoms. This allows the mapping of specific amino acid residues and their chemical environments.
Explain 1H-13C HSQC
heteronuclear NMR*
correlates proton (¹H) and carbon (¹³C) nuclei in a sample
The experiment begins by applying a radiofrequency (RF) pulse that excites both the ¹H and ¹³C nuclei in the sample. This pulse flips the nuclear spins of both the protons and the carbons, perturbing their equilibrium positions.
The x-axis corresponds to the ¹H chemical shifts.
The y-axis corresponds to the ¹³C chemical shifts
Each cross-peak in the 2D spectrum corresponds to a proton (¹H) that is directly bonded to a carbon (¹³C). The position of the peak in the x-axis corresponds to the proton chemical shift, and the position in the y-axis corresponds to the carbon chemical shift.
Diagonal Peaks:
The diagonal in the 2D spectrum represents where the proton and carbon chemical shifts correspond to the same nucleus (i.e., the diagonal represents the “reference” regions of the spectrum, but these peaks do not provide new information)..
What can INEPT be used for*
INEPT is a polarization transfer technique in NMR that enhances signals of insensitive (low-gamma) nuclei by transferring magnetization from a more sensitive nucleus, typically H (proton). It relies on J-coupling (scalar coupling) between the two nuclei.
A series of π (180°) pulses are applied to selectively transfer polarization from the proton to the heteronucleus.
This flips the less sensitive nucleus’s magnetization into the transverse plane, increasing signal intensity.
How does 3D NMR work*
How does 3D protein NMR work (HNCO)*
3D HNCO is a powerful NMR experiment used for backbone assignment of proteins. It correlates the amide proton , the amide nitrogen (
N), and the carbonyl carbon (
13
C
′
13
C
′
) of the previous residue in a protein sequence
Excitation of 1H ,The amide proton is excited first.
Transfer to 15N via INEPT
Polarization is transferred from 1 H to 15N
Transfer to 13 C via J-Coupling
Magnetization moves from amide nitrogen to the carbonyl carbon of the previous residue.
Reverse Transfer for Detection
The signal is transferred back to
, which is detected.
How does HN(CA)CO protein NMR work
As in HNCO, the experiment starts by exciting the amide proton.
Polarization Transfer to
15
N
15
N
Via INEPT (J-coupling transfer), magnetization is transferred to amide nitrogen.
Transfer Through
13
C
α
13
C
α
to
13
C
′
13
C
′
(Carbonyl Carbon)
Unlike HNCO, which transfers magnetization directly from
15
N
15
N to
13
C
′
13
C
′
, HN(CA)CO introduces an additional step:
Magnetization is transferred from
15
N
15
N to
13
C
α
13
C
α
of the same residue.
From
13
C
α
13
C
α
, it is then transferred to
13
C
′
13
C
′
of both the current and preceding residues.
Reverse Transfer and Detection
The magnetization is transferred back to
1
H
N
1
H
N
for detection
How does HN(CA)CB protein NMR work*
Excitation of
1
H
N
1
H
N
(Amide Proton)
The amide proton of the residue is first excited.
Polarization Transfer to
15
N
15
N
Via INEPT transfer, magnetization moves from
1
H
N
1
H
N
to
15
N
15
N.
Transfer Through
13
C
α
13
C
α
and
13
C
β
13
C
β
(Both Current & Previous Residues)
Magnetization is transferred from
15
N
15
N to
13
C
α
13
C
α
and
13
C
β
13
C
β
of both:
The current residue.
The preceding residue (via J-coupling through
13
C
α
13
C
α
).
Reverse Transfer and Detection
The signal is transferred back to
1
H
N
1
H
N
for detection.
How does HN(CO)CACB protein NMR work*
The amide proton of the residue is first excited.
Polarization Transfer to
15
N
15
N
Magnetization moves from
1
H
N
1
H
N
to
15
N
15
N via INEPT transfer.
Transfer Through
13
C
α
13
C
α
and
13
C
β
13
C
β
(Only Current Residue)
Unlike HN(CA)CB, which detects signals from both the current and previous residues, HN(CA)CACB restricts transfer to the same residue.
Reverse Transfer and Detection
The signal is transferred back to
1
H
N
1
H
N
for detection.
Explain the NMR structure calculation
How is NMR of IDPs done and analysed*
15
N R₁ and R₂ relaxation rates distinguish rigid vs. flexible regions.
High R₂/R₁ → More structured regions
Low R₂/R₁ → Highly flexible regions
Heteronuclear NOE (
1
H
−
15
N
1
H−
15
N) is weaker for disordered regions and stronger for folded domains.
Residual Dipolar Couplings (RDCs) and Paramagnetic Relaxation Enhancement (PREs)
RDCs reveal residual structural tendencies by measuring alignment in weakly ordered media.
PREs identify transient interactions and compaction by introducing paramagnetic spin labels.
IDPs show few NOE cross-peaks, reflecting weak long-range contacts.
IDPs often undergo conformational transitions upon binding to partners.
CSP mapping identifies residues affected by binding events.
Large chemical shift changes indicate induced folding upon binding.
Chemical shifts (
1
H
N
,
15
N
,
13
C
α
,
13
C
β
,
13
C
′
1
H
N
,
15
N,
13
C
α
,
13
C
β
,
13
C
′
) provide information about secondary structure propensities.
Random coil chemical shift values are used as a reference.
Deviations from random coil shifts (Secondary Chemical Shift Index, CSI) indicate transiently populated structures.
What are the differences between the high and low energy states of proteins
What is the E state of a protein composed of
Proteins are not static — they exist as dynamic ensembles of conformations. The E state represents a higher-energy, less-populated conformational state that a protein can transiently adopt, beyond its stable “ground state” (G state)
Proteins can switch between different shapes or conformations:
Ground state (G): Lowest energy, most populated, most stable.
Excited state (E): Higher energy, less populated, often short-lived.
Energy landscape:
Proteins have a rugged energy landscape with valleys (stable states) and hills (transition states).
The E state is one of the local minima, but not the global minimum.
Each local minimum represents a stable or semi-stable conformation.
The lowest minimum is usually the native (N) state — the most stable, most populated.
Excited states (E states) are higher-energy, less populated minima that may still play critical functional roles.
Fast motions explore local minima around a single conformational basin.
Slow motions allow the protein to jump between different minima — including visiting E states.
What does protein dynamics play a role in
What is meant by NMR relaxation
What is relaxation caused by
What is the process of NMRrelaxation for dynamics determination
What extra measurements are made in NMR relaxation for dynamics determination
What are heternuclear NOE measurements used for
How is the data interpreted for NMR relaxation for protein dynamics with the example of PKA
How do proteins attain biological activity
Explain the principles of chemical exchange in proteins
How can chemical exchange be seen on NMR
What does NMR with two state chemical exchange with 50% population of each state look like *
In two-state chemical exchange, a molecule interconverts between two conformations or binding states
When both states are equally populated (50% A, 50% B), the appearance of the NMR spectrum depends on the exchange rate (k
e
x
ex
) relative to the chemical shift difference (
Δ
ω
Δω) between states.
Slow Exchange (
k
e
x
≪
Δ
ω
k
ex
≪Δω)
The exchange rate is much slower than the chemical shift difference.
Two distinct peaks are observed at the chemical shifts of A and B.
Each peak has equal intensity (50:50 ratio).
Intermediate Exchange (
k
e
x
≈
Δ
ω
k
ex
≈Δω)
The exchange rate is similar to the shift difference.
Peaks broaden significantly and may merge into a single, broad, distorted peak.
This regime is often studied with CPMG relaxation dispersion experiments to extract exchange rates.
Fast Exchange (
k
e
x
≫
Δ
ω
k
ex
≫Δω)
The exchange is much faster than the shift difference.
A single peak appears at the weighted average chemical shift:
δ
obs
=
p
A
δ
A
+
p
B
δ
B
p
A
+
p
B
δ
obs
=
p
A
+p
B
p
A
δ
A
+p
B
δ
B
Since
p
A
=
p
B
=
50
%
p
A
=p
B
=50%, the peak appears at the midpoint between A and B.
Peak broadening depends on
k
e
x
k
ex
.
What does NMR of chemical exchange with skewed populations look like *
What is the exchange regime determined by
What is CPMG
How does a CPMG experiment work
What happens in a CPMG experiment if there is no exchange (so if there’s only one state)
What happens in a CPMG experiment if there are two states so chemical exchange occurs
Explain CPMG relaxation dispersion*
What parameters can be obtained from dispersion curves
It reveals dynamic processes where a nucleus (e.g. an amide proton) interconverts between two (or more) chemical environments with different chemical shifts
In CPMG relaxation dispersion NMR, we exploit the fact that:
If a nucleus is exchanging between two (or more) chemical environments (say A and B), its (R2) relaxation rate becomes dependent on the rate and extent of exchange.
By varying the frequency of refocusing pulses we can measure how the observed transverse relaxation rate, R2eff changes — this is called a dispersion curve.
Why It’s Useful
• Detects “invisible” excited states (low population, short-lived)
• Measures protein conformational dynamics, folding intermediates, binding kinetics
• Often used with 15N, 13C, or 1H NMR on backbone amides
How can NMR tell is differences between the two states of a protein with the example of T4 lysozyme
What can NMR tell us about state B of the protein (second state)
What can NMR experiments on protein dynamics tell us
What are the characteristics of ribonuclease A
What are the characteristics of dihydrofolate reductase
How was info on the states of dihydrofolate reductase obtained
What are the main points about CPMG
What are the limitations of studying IDPs
How can IDPs be recognised on 1H-15N HSQC spectrums
How can secondary structures in IDPs be seen using NMR
What is chemical shift anisotrophy *
What is the process of chemical shift anisotrophy
CSA refers to the fact that the chemical shift of a nucleus depends on the orientation of its local electronic environment relative to the magnetic field (B₀).
In other words, the magnetic shielding a nucleus experiences is direction-dependent — it’s anisotropic (not the same in all directions).
Why does CSA happen?
Nuclei are surrounded by electrons, which create tiny local magnetic fields when exposed to B₀.
These local fields shield the nucleus from B₀ to varying degrees depending on molecular orientation.
For non-spherical (anisotropic) electronic environments, the amount of shielding changes with direction → leading to different chemical shifts for different orientations.
Molecules tumble rapidly in solution, averaging the CSA effect.
The observed chemical shift is the isotropic average.
BUT: CSA still affects relaxation, especially T₂ (transverse) and T₁ (longitudinal) relaxation.
How is protein topology determined using paramagnetic relaxation enhancement
How was NMR used to determine an IDP region of the DNAJB6 protein
What’s the difference between tumbling of small and large molecules in solution
How does solid state NMR work
What is alpha synuclein
How can alpha synuclein be studied
What can CEST/DEST NMR be used for
CEST is used to detect low-population, invisible states that are in slow exchange with a visible, higher-population state.
How it works:
A selective radiofrequency pulse is applied at the frequency of the invisible or minor state.
If there’s exchange with a major state, the saturation transfers to the major state and causes a decrease in signal intensity.
By scanning the saturation frequency, you generate a CEST profile, revealing where the minor state exists and how it exchanges.
DEST is similar in principle to CEST but optimized for situations where the dark state is extremely broad and essentially invisible in the NMR spectrum (e.g., large aggregates or bound states with very fast transverse relaxation)
How DEST works:
A broad saturation pulse is applied to the region of the spectrum where the dark state would resonate.
Exchange with a visible, narrow-line free state transfers the saturation, reducing its signal.
DEST curves (plots of signal attenuation vs. saturation frequency) can be analyzed to extract information about the dark state.
How does chemical exchange in weak binding work
How does CEST or DEST NMR work
CEST detects low-population, “invisible” states (e.g., excited conformations, weakly bound ligands) that are in slow exchange with a major, visible state (usually on the milliseconds to seconds timescale).
Apply a weak, selective radiofrequency (RF) saturation pulse at a specific frequency — typically where the minor state would resonate.
If the minor state exchanges with the major state, the saturation is transferred to the major state over time.
This reduces the signal intensity of the major state.
By scanning the saturation frequency, you create a CEST profile — a plot of signal intensity vs saturation frequency.
Info obtained from CEST: exchange rate, population of the minor/invisible state, chemical shift difference between states, Structural/functional info about invisible states (e.g., conformational changes, binding events)
DEST is used when the minor (dark) state is extremely broad and invisible — such as aggregates, very large complexes, or strongly relaxing bound states.
From fitting the DEST profile, you can determine:
Exchange rates between free and dark (bound) states
Bound state population
Transverse relaxation rates of the dark state
Binding affinity and kinetics, even if the bound state is too broad to observe directly
What happens in NMR for proteins that form minor oligomeric / complex state in solution
Compare a CEST profile of a residue with / without exchange
An NMR method to probe exchange of NMR visible species with excited states. If the excited state has a large MW the method is also called DEST (dark exchange saturation transfer)
Based on the application of a weak saturating field (B1) at various places (offsets) on the spectrum
When an NMR resonance is saturated (ie when the B1 field is applied at tis frequency : offset ~0) its intensity is diminished (dark blue)
If there is exchange with another conformation that has a large MW saturation of the main observable species is observed at large offsets (light blue)
This happens because saturation of the large exited state is transferred to the small NMR observable main state through chemical exchange
How can the binding of alpha synuclein to liposomes be probed using CEST *
What is the function of alpha synuclein
How are alpha synuclein mutants with enhanced activity produced
What is a bad characteristic of alpha synuclein
How can the structure of toxic oligomers be elucidated by NMR
What are the applications of CEST and how does it work
What is the medical application of CEST MRI
What is gluCEST MRI
How can membrane proteins be studied using ssNMR*
Since membrane proteins are embedded in the lipid bilayer, they cannot be directly studied in solution NMR. Therefore, the proteins need to be reconstituted into model lipid bilayers (e.g., lipid vesicles, bicelles, or lipid nanodiscs) that mimic the natural membrane environment.
Solid-state NMR is uniquely suited for studying membrane proteins because it works with samples in the solid phase or non-crystalline states, making it applicable to proteins embedded in membranes or lipid bilayers.
High resolution is achieved by spinning the sample rapidly (at around 10-70 kHz) to average out anisotropic interactions like dipolar couplings and chemical shift anisotropy, which typically broaden NMR peaks.
Cross-polarization is used to transfer magnetization from abundant nuclei (like ¹H) to less abundant nuclei (like ¹³C or ¹⁵N), improving the sensitivity of the NMR experiment.
Membrane proteins have well-defined inter-atomic distances that can be measured using dipolar couplings (through the H-H, C-H, or N-H interactions)
