3 - NMR and Nascent Chain Folding

Question 1

What is nuclear spin?

Answer 1

All nuclei spin about their axis, creating a localised magnetic field. Usually the direction of the N-S axis of this field is random, and they all have equal energy.

Question 2

What effect does an external magnetic field have on the nuclear spins of the atoms?

Answer 2

when a strong external magnetic field is applied the magnetic axis sort into two states; they orient themselves either with the direction of the magnetic field or against it.

Question 3

When an external field is applied, how populated are the different spin states?

Answer 3

Aligning the axis with the external field is a lower energy state then aligning against it, though only slightly; at room temperature both are populated (according to the Boltzmann distribution) with only a slight bias towards alignment. However this bias is enough to give the sample a net magnetic moment.

Question 4

What is the Larmor frequency?

Answer 4

When in an external magnetic field, the N-S axis of the nuclear magnetic field precesses around the fixed direction of B0. The frequency of this precession is called the Larmor Frequency, and is determined by the gyromagnetic ratio; a constant for each different nucleus.

Question 5

What does the precession of the nuclei allow for?

Answer 5

The precession allows the nuclei to resonate with electromagnetic radiation when the frequency of the wave is equal to the Larmor frequency. This puts it in the radio frequency range; 10-1000 MHz. The machine being used must be tuned to the frequency of the nuclei in question.

Question 6

What does the frequency of an NMR machine describe?

Answer 6

The higher the maximum frequency of the NMR machine the higher the sensitivity and resolution of the resulting data.

Most NMR machines operate at 500-700MHz, and are capable of looking at multiple nuclei of a biological sample. The 800MHz and the 950MHz machines are capable of being used for high-throughput metabolomics studies.

Question 7

How can nuclear spin be thought of quantum mechanically?

Answer 7

Nuclear spin is a quantum mechanical property of the quarks within the nucleus, hence why the total spin number of the nucleus is dependent on the baryon combination that constructs it. The total spin of the element/isotope (I) can be any number that is a multiple of ½ - 0, ½, 1, 1½, 2, 2½, etc.

Question 8

What nuclei are NMR active?

Answer 8

Only nuclei with odd mass numbers are NMR active, with the exception of 2H and 15N, but the most common nuclei in NMR are 1H and 13C which are both I = ½ (‘spin-half’ nuclei).

The total number of spin states = 2I + 1, so these have 2 spin states; up and down.

Question 9

How is the sample loaded into an NMR machine?

Answer 9

When the sample is within the magnetic field produced by the superconducting (and super-cooled – 4.2K) magnets it is spun to ensure field homogeneity.

Question 10

What happens when a sample is probed by radio waves?

Answer 10

The radio transmitter and receiver are tuned to the Larmor frequency in question and the transmitter saturates the sample, flipping the spin states of the nuclei to the higher energy level.

Question 11

What is measured in NMR spectroscopy?

Answer 11

The change in the effective energy gap between the spin states, and hence the radio frequency at which they absorb and emit. This will be different for different nuclei due to their electronic environment.

Question 12

What is electronic shielding?

Answer 12

Electrons themselves also spin, usually in a way that opposes the external magnetic field. This reduces the effective B0 upon the nucleus. Hence the larger the decrease in the energy separation the more electrons are in the area of that nucleus.

Question 13

How is electronic shielding of the nuclei measured?

Answer 13

This must be measured relative to a known structure, often TMS, which is included with the sample.

Question 14

How can measurements of shielding build up a picture of the molecule?

Answer 14

Since the strength of the signal is proportional to the population of nuclei in the same electronic environment, a picture can be built up of the molecule being imaged.

The variation in RF over time can be converted into a spectrum of populated frequencies through a Fourier transform.

Question 15

What else can effect the difference in energy between the spin states?

Answer 15

The difference in spin energy levels between a nucleus’s spin state is also dependent on the spin state of the nuclei around it. This is called spin-coupling; when an adjacent nucleus is in the spin up state the nucleus in question will have a slightly different energy of absorption then when the adjacent nucleus is in ‘spin down’.

Question 16

What does spin coupling do to the spectrum?

Answer 16

This splits the peak, and allows identification of the atoms adjacent to each nucleus. This allows us to use the scalar and dipolar couplings to match the set of signals with a molecular structure. This is called Resonance Assignment.

Question 17

What is scalar and dipolar coupling?

Answer 17

Scalar coupling is though bonds, position of nuclei relative to one another constant-ish.

Dipolar coupling though space - not connected by bonds.

Question 18

What is the nuclear overhauser effect.

Answer 18

Coupling through space as opposed to through adjacent bonds is a phenomenon known as the Nuclear Overhauser Effect (NOE). This can be used to analyse relative positions of atoms up to 5Å apart.

Question 19

What does the 1D spectrum of a protein look like?

Answer 19

Being huge molecules, proteins produce hundreds of thousands of signals and resonance assignment becomes very difficult. Very little structural information can be derived from 1D NMR.

To get around this issue, 2D NMR can be used.

Question 20

What is 2D NMR?

Answer 20

This uses series of pulses and recordings to add extra time dimensions, each one producing another frequency axis. Various different pulse combination protocols have been devised, such as NOESY and DANTE. These often use the NOE to analyse the interactions between long-range tertiary structural features.

Question 21

What are the limitations of 2D NMR?

Answer 21

This gives a limit to the size of the protein in question of 100-120 amino acids. Any more than this and the spectrum becomes too crowded.

Question 22

What is the best way to determine protein structure using NMR?

Answer 22

Heteronuclear Single Quantum Coherence (HSQC) spectroscopy

Question 23

What is HSQC?

Answer 23

This uses multiple types of NMR active nuclei on the same protein, often using 15N – 1H pairs on the backbone but also sometimes using carbon as a third axis.

This allows each residue to be plotted as a point on a pair of axis of the chemical shift of each of the heteronuclei, thus splitting up the peaks.

Question 24

How can HSQC show the foldedness of a protein?

Answer 24

More folded proteins have higher dispersion as the different parts of the protein can affect the electronic environment of each residue, while disordered proteins show poor dispersion as most residues have similar environment being mostly surrounded by water.

Question 25

What is one of the main challenges of HSQC NMR?

Answer 25

Assigning signals from the fingerprint regions of the spectra to specific residues can be difficult. The goal is to identify the resonant frequencies of all hydrogen, carbon and nitrogen nuclei in a protein to determine the structure.

Question 26

What techniques allow for resonance assignment in HSQC NMR?

Answer 26

The methods of doing this can be classified as backbone assignment strategies or side-chain assignment strategies. These use analysis of different combinations of the atoms to deduce which signal is from which. One of the most important is the HNCA/HN(CO)CA backbone assignment strategy.

Question 27

What is different about structures produced by NMR rather than XRC or CEM?

Answer 27

Unlike crystallography or electron microscopy, NMR does not produce an experimental structure of the protein. Instead, NMR yields distance restraints which can be used to calculate presumed structure. Additionally NMR allows us to view dynamic, native structures, unlike XCR.

Question 28

How are NMR distance restraints used to make a protein structure?

Answer 28

These structure calculations yield multiple solutions (ensembles) to the distances between the atoms, so the final NMR protein structures are always ensemble averages.

Question 29

How is secondary structure identified in HSQC NMR?

Answer 29

It is the carbon chemical shift values that provide the secondary structure. This is done by comparing the random Cα and Cβ chemical shift values to those that are found then they form a coil or sheet.

Question 30

What is Residual Dipolar Coupling used for?

Answer 30

This is a powerful and rapid method of determining protein structure using dipolar coupling. RDC is used to improve the quality of NMR structures, and can greatly improve the resolution of the structures.

Question 31

How does Residual Dipolar Coupling work?

Answer 31

Analysis of the level of dipolar coupling between two bond-linked atoms can provide information about the orientation of that bond relative to the magnetic field. If you can calculate the coupling constant (D) for each bond pair then an entire structure can be derived.

Question 32

What is the challenge associated with Residual Dipolar Coupling structural determination?

Answer 32

In solution the signal for each bond will average to zero due to isotropic tumbling; the proteins must be aligned in order for the orientations of their bonds to not just produce noise.

Question 33

How is the problem of isotropic tumbling overcome to enable Residual Dipolar Coupling structural determination?

Answer 33

Isotropic tumbling can be removed by orienting the proteins in the same direction using bicelles, virus particles or polyacrylamide gels. These are designed to prevent random tumbling of the protein through mechanical impediment, but the interaction between the protein and the alignment medium can create issues.

Question 34

What is the primary advantage of NMR over XRC or EM?

Answer 34

The dynamic nature of NMR spectroscopy is one of its greatest assets. It allows analysis of protein motion and hence conformation change events, folding and unfolding and ligand binding.

Question 35

How can NMR monitor ligand binding events?

Answer 35

By seeing which residues have a shifted chemical shift before and after the binding event, describing the change in electronic environment.

Question 36

What can NMR analysis of ligand binding be used for?

Answer 36

NMR study of binding events is site-specific, allows for multiple probes and gives in-depth information including spatial responses. This makes it very easy to determine the Kd of a ligand binding event, which is very useful for analysis of drug binding studies.

Question 37

Which techniques or parts of the results of an NMR spectroscopy experiment report on dynamics?

Answer 37

``` Linewidths H-D exchange NMR relaxation measurements NOE T1 spin lattice relaxations T2 spin-spin events ```

Question 38

How do linewidth changes report on dynamics?

Answer 38

Linewidths will change depending on the motion of the protein. Narrower line-widths indicate faster motion and wider linewidths slower. This is dependent on molecular weight or conformational change.

Question 39

What is H-D exchange used for?

Answer 39

H-D exchange can be used to track the change in solvent exposure, analysing local or global unfolding events that take place over slow timescales.

Question 40

In what timescale can NMR relaxation measurements report on dynamics?

Answer 40

NMR relaxation measurements can track motion in the ps-ns and ms range.

Question 41

In what timescale can T1 spin lattice relaxation report on dynamics?

Answer 41

T1 spin lattice relaxation reports on fast motions.

Question 42

In what timescale can T2 spin-spin events and the NOE report on dynamics?

Answer 42

Fast or slow motions

Question 43

What is cotranslational folding?

Answer 43

Cotranslational folding is the way in which proteins fold as they sequentially and in a vectorial manner (i.e. N-C) emerge from the ribosomal exit tunnel, which is thought may sometimes have large effects on the folding of a protein.

Question 44

Why is cotranslational folding thought to alter the way in which proteins fold?

Answer 44

The tethering and slow emergence (compared to the rate of folding events) mean that there is a different folding landscape for nascent proteins, causing it to sample different conformations and populate different intermediates.

Question 45

What did nascent chain folding studies show about luciferase?

Answer 45

Luciferase is a 62kDa multi-domain protein that, when denatures in vitro will not refold once the denaturants are removed, instead forming a catalytically inactive aggregate. This is because when translated there is early formation of a stable folding intermediate in form of a 190 residue domain around which the rest of the protein folds.

Question 46

How is cotranslational folding imaged?

Answer 46

While EM can be used to determine the structure of the chain inside the tunnel, non-dynamic structural studies cannot be used to visualise the structure of the emerging chain do to its constant random sampling. NMR is more suited for this, but it is a difficult process.

Question 47

What provides proof of concept for the use of NMR in analysis of cotranslational folding?

Answer 47

NMR has been used to characterise the dynamic stalk subunit of the ribosome by using an N-15 labelled structure. This showed that L7 visited various parts of the ribosome during the translation cycle.

Question 48

How are cotranslational folding studies using immunoglobulin performed?

Answer 48

Immunoglobulin is often used as a model for nascent chain studies. Folding snapshots can be created by introducing empty codons or SecM into the reaction to stall the ribosome.