NMR Flashcards
What are the pros and cons of biological NMR
pros:
-provides both structure and dynamics
-no need to crystallise sample
-probe molecular interactions
-near physiological conditions (can study protein in solution/even in cell)
cons:
-size limit (35kDa)
-requires isotopic labelling
What information can you get from NMR spectra
- Chemical shift (δ) – chemical environment
- Integral (area under peak) – number of equivalent spins
- Scalar coupling (J) – local structure
- Relaxation times (T1 and T2) – dynamics
- Dipolar coupling (D) – long range structure
- Nuclear Overhauser effect (NOE) – structure/binding
Describe the process of performing NMR
- Start with having free protons (all in random spins)
- Apply magnetic field (upward spin, z-axis)
- Proton spins will align w/ external MF - population of protons with upward spins exceed downward spins, so eq/net magnetization points upwards – in z axis
BUT detector is located in X/Y plane - Radiofrequency pulse applied along y axis (90° to the spins) causes the z magnetization to be rotated onto x axis (transverse magnetization)
- Measure how the magnetization precesses & decays along the XY plane to restore its z magnetization
* Measured as Free induction decay in time domain - FT to frequency domain - observe chem. shift peaks
* FID of each proton = depend on the environment they’re in = diff. chem. shift
What are the problems with larger molecules
- FID of 2 closely spaced peaks is only distinguishable if they take a long time to phase out.
- If the signal decays after a short period of time, there will only be 1 broad average peak of the 2 protons observed
Explain what occurs in 2D NMR
- A second RF (90°) pulse is generated after a certain amount of time delayed from the first RF pulse.
- The delayed period involves the evolution of magnetization along an indirect dimension.
- After the second RF pulse, an additional mixing period is required for the mix/ transfer of signals between coupled spins.
- The FID after the second pulse is recorded in t2 and then detected to obtain 2D spectra.
For each point to be obtained on the 2D spectra, the whole experiment must be repeated multiple times by varying the evolution time (between pulse 1 & 2) - time consuming
What generates a set of FIDs in the time domain
different evolution times:
- FT rows for frequencies on x axis
- FT columns for frequencies on y axis
- Result in 2D NMR peaks
What are the types of 2D NMR
Homonuclear NMR (observe chemical shifts of the same species eg. H-H)
Heteronuclear NMR (observe chemical shifts of 2 different species eg. N-H)
Explain heteronuclear NMR
- first excitation is followed by an INEPT element to enable the transfer of the excitation from the H to N
- After the evolution time, the polarization is transferred back to H via another INEPT element.
- Like homonuclear 2D NMR, detection of FID is made during t2
What is the criteria for a good nucleus for recording heteronuclear NMR
- Spin ½, high abundance, high magnetogyric ratio = high sensitivity
- For C & N, abundance is low – must increase sensitivity: growing bacteria in medium containing the labelled isotope
o For 13C, use 13C labelled glucose
o For 15N, use 15N labelled ammonium chloride - The labelled isotopes will be included in the POI produced in the bacteria
- Supplements of vitamins and trace elements of minerals required for the bacteria to grow in the unnatural media
Describe H-N HSQC heteronuclear spectra
- represents scalar coupling across N-H bond, so only amine groups will appear as a peak, corresponding to 1 aa
- side chains containing NH groups (ex. glutamine, asparagine) will also appear as a peak
How is 3D NMR performed
- 3 RF pulses applied and record FID in t3
- Can be heteronuclear NMR – measure frequencies of 3 different nuclei eg H, N, C Or H, H, N
What are the 4 popular 3D NMR experiments:
HNCO
HNCACO
HNCACB
HNCOCACB
Describe HNCO
- Magnetization is passed from 1H to 15N and then selectively to the carbonyl 13CO
- Magnetization is then passed back via 15N to 1H for detection.
- CO i-1 is detected
> in each NH strip, there is one CO visible which belongs to the preceding residue
Describe HNCACO
- Magnetization is transferred from 1H to 15N and then to the 13Cα and to the 13CO
- For detection the magnetization is transferred back from 13CO to 13Cα, 15N and finally 1H
- Because the amide NH is coupled to both Cα of its own residue and of the preceding residue, transfers to both 13CO nuclei occurs
- For each NH group, 2 CO groups are observed in the spectrum. because the coupling between Ni and Cαi is stronger than that between Ni and Cαi-1, the Hi-Ni-COi peak generally ends up being more intense than the Hi-Ni-COi-1 peak.
Describe HNCACB
- Magnetisation is transferred from 1Hα and 1Hβ to 13Cα and 13Cβ, respectively, and then from 13Cβ to 13Cα. From here it is transferred first to NH and then to HN for detection.
- magnetisation is transferred to 15Ni from both 13Cαi and 13Cαi-1.
- Thus for each NH group there are two Cα and Cβ peaks visible (for same and preceding residues)
Describe HNCOCACB
- Magnetisation is transferred from 1Hα and 1Hβ to 13Cα and 13Cβ, respectively, and then from 13Cβ to 13Cα. From here it is transferred first to 13CO, then to 15NH and then to 1HN for detection.
- The chemical shift is evolved simultaneously on 13Cαi-1 and 13Cβi-1
How are 3D NMR usually used
usually used in combination with each other
eg
* An overlay of the HNCO and HN(CA)CO spectra makes it very easy to distinguish between COi and COi-1 for each NH group.
if the peak occurs both in HNCO and in HNCACO, it belongs to preceding residue CO.
If peak occurs only in HNCACO, belongs to same residue CO.
How to calculate NMR structure
- Utilizes Simulated Annealing (SA)
1. starting structure is heated in a simulation (i.e. the atoms of the starting structure get a high thermal mobility) and then cooled
2. During cooling steps, the starting structure can evolve towards the energetically favorable final structure under the influence of the force field - The force fields involve contributions from input restraints to guide folding of proteins - ideal bond length, ideal bond angles, including information derived from NMR – the NOE-derived distance bounds, and coupling-constant-derived dihedral angle restraints.
Etotal=Ebond + Eangle + Evdw + Edihedral + Enoe
Since there could be multiple ways to satisfy the input restraints, the calculation is repeated many times (>100) to generate an ensemble of structures. If enough/ correct restraint is given in the input, the structures should be similar but not identical.
What can you use NMR for
- Determine weak protein-protein interactions
o β2m forms dimers
o obtain NMR spectrum of β2m dimers at different concentrations – each conc. = diff chem shifts
o from overlaying diff. spectra can fit a binding curve to determine the weak dimerization constant - Structural approaches to IDPs
o Techniques such as X-ray crystallography are limited in the study of IDPs
o IDPs are virtually impossible to crystalize & even by crystallizing an IDP, the resulting structure would represent only one of the infinite conformations that the protein can adopt
*Disordered proteins can be immediately recognized in a 1H-15N HSQC spectrum for two characteristics:
o limited dispersion (Backbone 1HN restricted in ~1ppm)
o sharp peaks (long T2)
- Thus, even if the spectra are poorly dispersed, the resonances are normally well resolved and present limited overlap
Describe high and low energy states of proteins
- Low energy states of proteins = highly populated & can be studied easily
- High energy states of proteins (carry biological function) = less populated & can only be studied with some NMR techniques
- Protein dynamics involved in achieving the high energy states = also contribute to the function
Explain protein dynamics
- Each E state of a protein is composed of a number of local minima
- Diff E state, and diff local minima in 1 E state, are all divided by E barriers – but of different sizes
E barrier between diff. E states = higher barrier, thus result in slower dynamics (µs-ms) and are associated with large conformational changes
E barrier between diff local minima in 1 E state = low barrier, thus result in faster dynamics (ns-ps) and are associated with smaller conformational changes (could just be vibrations in atoms/ bonds, etc.)
How do proteins use dynamics to perform their functions
- Protein dynamics play a role in protein-protein interactions
- also allow for conformational flexibility, giving rise to 2 major properties:
* Biomolecular promiscuity: majority of proteins have more than 1 interacting partner
* IDPs ability to adopt an infinite number of conf. allow it to function as a hub in a network of PPI - Protein dynamics is also related to enzymatic activity
* Enzymes = not functional in lower temperature due to restricted motion
Explain NMR relaxation
Relaxation is the process by which the spins return to equilibrium
- Longitudinal relaxation (T1) – restoration of Mz at a rate of R1
- Transverse relaxation (T2) – fading of Mxy at a rate of R2
Small proteins:
short T1, long T2
Large proteins:
long T1, short T2
Relaxation is caused by time-dependent fluctuations in the local MF – brought about by protein motion/ protein dynamics
* Short T2 relaxation (fast decay of signal) = broad peaks – which is why NMR is better for small proteins
Describe the process of NMR relaxation for dynamics determination
- sample preparation (eg. prepare isotope source, etc)
- assignment of protein spectra (assign each peak to each residue)
- carry out dynamics measurements
- interpret results
What is the most popular type of relaxation
- 15N relaxation is the most popular, it infers on N-H
Explain R1 and R2 measurements
In addition to the 2D heteronuclear NMR method of having INEPT elements after the pulses to enable the transfer of the excitation between nucleus, a new block of pulses is added before the second INEPT element -time for relaxation delay.
Different values of τ give different signal intensities – shorter delay = more intense, but longer delay = less intense, so can form multiple HSQC, can observe decay in signal intensities
The pulse sequences are made in a way that the decay of signal intensities as a function of τ are proportional to either R1 or R2
We fit these decays with single exponential function and obtain R1 or R2 for every peak (hence every residue) in the HSQC.
Explain heteronuclear NOE measurements (cross-correlated relaxation)
- The backbone 1H-15N heteronuclear NOE provides information about the motion of individual N-H bond vectors
- Perform 2 experiments: 1 normal HSQC & 1 where H is saturated to measure the effects on N
- Ratio of signal intensities between the 2 experiments = hetNOE
o Rigid NH vector = NOEHX ~ 0.9
o Dynamic NH vector = NOEHX «_space;1
Describe the data interpretation + example
The info. obtained can be used to calculate order parameters –
S2: magnitude of angular fluctuation for chemical bond vector (modelled as theta angle describing how much the bond vector moves in a cone)
S2 = 1 (rigid motion), S2 = 0 (isotropic motion)
Ex. PKA
* has 2 domains – small & large lobe
* interchangeably in open & closed conformation, depending on binding of substrate
* N relaxation experiment performed – can obtain R1, R2, hetNOE value at diff states of proteins (ex. when unbound, when nucleotide-bound, when substrate-bound)
* Shows that protein is very dynamic in all the states
* Can also observe dynamics of the substrate
