Modern approaches to protein

Question 1

What does the gyromagnetic ratio determine

Answer 1

The gyromagnetic ratio determines the ratio of nuclear magnetic moment to nuclear spin

Question 2

What do fundamental symmetry theorems predict

Answer 2

Fundamental symmetry theorems predict co-linear spin and magnetic moment

Question 3

What is the equation for magnetic moment

Answer 3

u=gamma x I

How much magnetism (u) we get for spin I

Question 4

What does energy depend on

Answer 4

size and relative orientation of B and u

Question 5

What is Zeeman splitting

Answer 5

delta E = gamma hbar B0

split by different values of m

Question 6

What is the Larmor frequency

Answer 6

Resonance corresponding to the Zeeman splitting induced in NMR experiments
w0 = gamma B0

Question 7

Why do we need a large magnetic field

Answer 7

Boltzmann distribution gives 1 in 1000 nuclei with spin a over spin B; a and B populate according to Boltzmann. Larger delta E scale with B0; larger population difference gives more nuclear magnetisation and a larger magnet increases the population therefore is good for experiments

Question 8

Give the protocol of a 1D FT NMR experiment

Answer 8

1) There is a slight population excess in lower energy alpha which leads to net magnetisation along z and no net in xy as nuclei are precessing in B0 therefore randomised and no net magnetisation
2) Generation of transverse magnetisation by 90* pulse, perpendicular to magnetic field
3) Place receiver coil perpendicular to magnetic field with a pre amplifier and detect oscillating magnetic moment as it induces small electric signal on the order of nanoV
—> NMR signal also called free induction decay FID
4) FID: transverse magnetisation oscillates and decays
Rate of decay in x: transverse relaxation time
Rate of decay in y: spin-spin relaxation time

Question 9

Why do we run experiments in D2O

Answer 9

Doesn’t have net spin so doesn’t show up in spectra; labile protons (amides) don’t show up as well. This clears up NMR spectra

Question 10

How do we use pH to monitor a peak for example which protons are bound to an active site

Answer 10

Can measure the pKa because shifts decrease on increasing pH and can plot a sigmoid curve: pKa at 50% complete change
Then repeat titrations in presence of an inhibitor and the ones that still change are protonated in the enzyme-inhibitor complex; the ones that are protonated in the complex are protected from exchange and therefore a higher pH is required to remove them

Question 11

What are the 3 stages to an NMR experiment

Answer 11

resolve resonances, assign, interpret data

Question 12

What is COSY

Answer 12

Correlation spectroscopy: you get cross peaks from transfer of magnetisation
Scalar J coupling means you see transfer from protons within amino acid side chains and amide proton and alpha carbon proton

Question 13

Typically whats the J limit you can see in COSY

Answer 13

3J: you do not see across the peptide bond

Question 14

What is a spin system

Answer 14

A spin system arises from scalar coupling within an individual amino acid with a carbonyl as a spacer because you do not see coupling across the peptide bond

Question 15

What do you see in the 0-5 ppm region of a COSY

Answer 15

amino acid side chain spin systems sitting on top of each other

Question 16

What is the fingerprint region of a COSY

Answer 16

Calpha - NH region;
top axis: 6.5 - 9 ppm
sideaxis: 3.5 - 5 ppm

Question 17

Where do you see Aromatic-aliphatic peaks in a COSY

Answer 17

top axis: 6.5 - 8.5 ppm

side axis: 0 - 2.5 ppm

Question 18

Why can ambiguity arise from a COSY spectrum

Answer 18

Say there are two alanine resonances with different shifts for NH and Me but degenerate alpha C proton shifts. You cannot link the NH to the correct side chain because they will appear at degenerate points on the spectrum

Question 19

What is TOCSY

Answer 19

Total correlation spectroscopy: produces cross peaks between all protons of a spin system and is therefore ideal for proteins

Question 20

What is a spin locking field

Answer 20

A spin locking field is a series of rapid 90* pulses of varying phase

• it averages proton-proton coupling constants over the entire spin system
• Magnetisation is transferred very efficiently at a rate determined by J
• Longer mixing time = magnetisation propagates further through the system
• Very small J can still cause cross peak

Question 21

How come we can correlate along the entire system in TOCSY

Answer 21

Dispersion of the NH-aH region

Question 22

What does NOESY allow

Answer 22

It provides data on internuclear distances and therefore you can correlate directly with molecular structure

Question 23

What is an nOe caused by

Answer 23

Cross relaxation: think of that energy level diagram with aa bb ab ba W2 is aa bb and W0 is ab ba

Question 24

In small molecules which is the dominant NOE effect

Answer 24

W2 because of rapid tumbling

Question 25

In large molecules which is the dominant NOE effect

Answer 25

W0: negative NOE enhancement W0 connects energy levels of similar energy so only low frequency required "cross relaxation"

Question 26

How do you carry out a 2D NOESY experiment

Answer 26

add additional mixing time to the COSY experiment and see a build up of magnetisation from one nucleus to a close neighbour

Question 27

Describe the protocol of a 2D NOESY experiment

Answer 27

``` Presaturation time 90* pulse t1 90* pulse: magnetisation of interest lies on -Z, this pulse means that cross-relaxation now occurs to a nearby nucleus mixing time 90* pulse t2 ~~~~~~~--- ```

Question 28

How do you work out the separation from NOESY

Answer 28

NOE = k (1/r6) so calibrate with known distance where k is a proportionality constant Detect up to 5A

Question 29

How do you use NOESY for sequential walking

Answer 29

See additional TOCSY peaks compared to COSY in the fingerprint of 2D NOESY from alpha proton to the NH proton of the next spin system Also see NHi - NHi+1 contacts which give additional info

Question 30

What is the definition of a short range NOE

Answer 30

less than 5 residues apart: used to detect alpha helix

Question 31

What is a long range NOE

Answer 31

more than 5 residues apart

Question 32

What is the characteristic NOE of an alpha helix (3 strong, 1 weak)

Answer 32

aHi - NHi+3 and aHi - NHi+4 due to the shape ~ 3.6 aa in a turn of a helix strong NHi - NHi+1 weak aHi - NHi+1

Question 33

What is the characteristic NOE of a beta sheet

Answer 33

aHi - NHi+1 and long range NOE. This is always very strong

Question 34

How do you distinguish an alpha helix from a 310 helix

Answer 34

No CaHi - NHi+2

Question 35

Define weak, medium and strong cross peaks

Answer 35

weak 1.8 - 5A medium 1.8 - 3.5A strong 1.8 - 2.5A Weak NOE does not mean large R; signal could have attenuated

Question 36

What is simulated annealing

Answer 36

Simulated annealing is a computer molecular dynamics and minimisation routine which uses the distance constraints from an NOESY spectrum to obtain a model of the structure. The programs contain info on amino acids, bond lengths and angles, standard interactions

Question 37

What is the protocol of simulated annealing

Answer 37

1) Simulate 1000K bath. Generate model strand along x (randomised y z) START 2) Apply distance restraints from NOE (~1000 for protein with 90 amino acids). Weight to favour formation of secondary structure and later long range strucutural features. Allow the chain to move through itself and reduce Van der Waals radii 30 PS 3) Start to cool the system and increase the penalty for not satisfying an NOE 20 PS 4) Minimise final structure to see if it satisfies all NOEs and unfold with secondary structure beginning to emerge 50 PS 5) 3D structure, hydrophobic groups packaged

Question 38

What are the challenges for interpreting 3D structures (2)

Answer 38

- Uncertainty must be shown for molecule in order to represent a structure not a model - polypeptides are dynamic therefore there is more than one conformation...which is biologically relevant?

Question 39

In which parts of a molecular structure do you see poor definition in NMR experiments

Answer 39

Loops and termini: flexibility for binding and function

Question 40

How do we work out the precision of an NMR determined structure

Answer 40

coordinate root mean square displacement vs average structure

Question 41

How do we work out the accuracy of an NMR determined structure

Answer 41

rmsd vs "true" structure

Question 42

How do we reduce the complexity of NMR experiments

Answer 42

use 15N as it is a spin 1/2 nucleus

Question 43

How do we label proteins with 15N

Answer 43

Give a bacteria 15NH4Cl and feed the plasmids to get bacteria to switch on specific genes and grow the proteins we want and then those proteins will be 15N labelled

Question 44

What is HSQC and why is it good

Answer 44

Heteronuclear Single Quantum Coherence 2 1H axes and a 15N axis. The 15N axis is very good at dispersing signals You get a correlation map and ambiguous peaks become resolvable. Can also do this with 13C

Question 45

How would you go about getting 13C proteins

Answer 45

Starve a bacteria system of all C other than 13C glucose Feed plasmids to grow the proteins we want High intensity 15N 13C 1H peaks!!

Question 46

Why do you need triple resonance experiments (why is NOESY not enough)

Answer 46

2D NOESY overwhelms space on the correlation map

Question 47

Which region of the 2D map can 15N simplify and which region can it not simplify

Answer 47

Can simplify the fingerprint region but not the aliphatic region

Question 48

What is the benefit of having 13C

Answer 48

Spin active 13C=O allows correlation between amino acids so no longer acts as a brick wall

Question 49

Describe stages in a triple resonance experiment

Answer 49

1) backbone assignments via 1J couplings - HNCA out and back from NH to aC-1 and aC - HN(CO)CA correlates N and NH of residue with aC of previous residue; doesn't record CO but uses it to correlate aC(i-1) with 1H and 15N of i 2) side chain assignment with HCCH-COSY and HCCH-TOCSY - HCCH: aliphatic side chains of individual amino acids: backbone assignments correlated with side chains (we know 13Ca shifts from stage 1) 3) 15N and 13C edited NOESY to resolve uncertainties in NOE

Question 50

Give the 5 stages of SAR by NMR for drug research

Answer 50

1) screen for first ligand 2) optimise first ligand 3) screen for second ligand 4) optimise second ligand 5) link ligands - --> high affinity ligands (nM affinity ligands from 2 uM affinity ligands)

Question 51

Why is SAR by NMR useful

Answer 51

Reduces discovery and synthesis time | Good for target directed synthesis

Question 52

What are the two mechanisms of spin relaxation

Answer 52

- Longitudinal or spin-lattic relaxation (T1 time constant) | - Transverse or spin-spin relaxation (T2 time constant)

Question 53

What is longitudinal or spin-lattice relaxation

Answer 53

Recovery of longitudinal magnetisation and establishment of thermal equilibrium populations with an exchange of energy Molecular tumbling - dipole of 1 nucleus creates oscillating field felt by neighbours

Question 54

What is transverse or spin-spin relaxation

Answer 54

Decay of transverse magnetisation No exchange of energy and does not restore system to equilibrium Loss of coherent transverse magnetisation with an increase in entropy ~~~~~~~~~---

Question 55

What is the Fourier Transform

Answer 55

The fourier transform is a computational method for analysing frequencies in an oscillating signal

Question 56

Why doesn't a normal experiment to measure T2 work well

Answer 56

Poor shimming and field inhomogeneity: field dephases due to inhomogeneity during tau/2

Question 57

How does spin-echo for measuring T2 work

Answer 57

Each magnetisation vector is rotated about y by a second pulse then they reconverge = echo

Question 58

What are the two mechanisms by which a system returns to equilibrium through spin-lattice relaxation

Answer 58

- Intrinsic process: D-D coupling between magnetic dipoles plays dominant role in relaxation of most spin 1/2 nuclei - Anisotropic electron density surrounding nucleus: chemical shift anisotropy- important in environments such as N and C

Question 59

What are the 3 mechanisms by which transverse relaxation occurs

Answer 59

Loss of coherence - inhomogeneous magnetic field: precessional frequency of spins differs based on location; nothing to do with intrinsic relaxation properties therefore is a nuisance - DD coupling 3 - Chemical shift anisotropy

Question 60

What is T1 affected by

Answer 60

fluctuations perpendicular to B0

Question 61

What is T2 affected by

Answer 61

fields along z

Question 62

How do we observe protein dynamics

Answer 62

By relaxation studies in the solution state Measure T1, T2 and NOE to 1H of amide and relate the 3 parameters to drive local and global motions and orientational order

Question 63

What is the timescale of overall motion of a protein

Answer 63

nanoseconds

Question 64

What is the timescale of motion of flexibility

Answer 64

picoseconds

Question 65

What is the problem with high molecular weight compounds

Answer 65

slower tumbling leads to fast decay which gives poor signal to noise

Question 66

Why do we use deuterium labelling for large molecules

Answer 66

For molecules above 25kDa 2D labelling reduces the relaxation (gD/gH)~1/0.5 which improves the signal to noise ratio for better resolution

Question 67

Which n-bonded protons do you see in a deuterium labelled experiment

Answer 67

Just the non-labile N-1H so on 15N you just see N-1H protons which makes a better spectrum

Question 68

What is TROSY

Answer 68

Transverse Relaxation Optimised Spectroscopy Switch decoupler on and off: 1J N-H = 90Hz Each peak of the multiplex relaxes at a different rate due to interference between relaxation mechanisms

Question 69

What components are there to relaxation, how do they appear in a spectrum

Answer 69

Fast relaxation components appear broad | and slow relaxation components appear sharp

Question 70

Which component does the pulse sequence select

Answer 70

Just the fast component