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Organic > Modern approaches to protein > Flashcards

Modern approaches to protein Flashcards

1
Q

What does the gyromagnetic ratio determine

A

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

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2
Q

What do fundamental symmetry theorems predict

A

Fundamental symmetry theorems predict co-linear spin and magnetic moment

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3
Q

What is the equation for magnetic moment

A

u=gamma x I

How much magnetism (u) we get for spin I

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4
Q

What does energy depend on

A

size and relative orientation of B and u

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5
Q

What is Zeeman splitting

A

delta E = gamma hbar B0

split by different values of m

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6
Q

What is the Larmor frequency

A

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

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7
Q

Why do we need a large magnetic field

A

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

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8
Q

Give the protocol of a 1D FT NMR experiment

A

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

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9
Q

Why do we run experiments in D2O

A

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

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10
Q

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

A

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

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11
Q

What are the 3 stages to an NMR experiment

A

resolve resonances, assign, interpret data

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12
Q

What is COSY

A

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

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13
Q

Typically whats the J limit you can see in COSY

A

3J: you do not see across the peptide bond

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14
Q

What is a spin system

A

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

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15
Q

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

A

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

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16
Q

What is the fingerprint region of a COSY

A

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

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17
Q

Where do you see Aromatic-aliphatic peaks in a COSY

A

top axis: 6.5 - 8.5 ppm

side axis: 0 - 2.5 ppm

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18
Q

Why can ambiguity arise from a COSY spectrum

A

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

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19
Q

What is TOCSY

A

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

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20
Q

What is a spin locking field

A

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
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21
Q

How come we can correlate along the entire system in TOCSY

A

Dispersion of the NH-aH region

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22
Q

What does NOESY allow

A

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

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23
Q

What is an nOe caused by

A

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

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24
Q

In small molecules which is the dominant NOE effect

A

W2 because of rapid tumbling

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25
Q

In large molecules which is the dominant NOE effect

A

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

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26
Q

How do you carry out a 2D NOESY experiment

A

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

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27
Q

Describe the protocol of a 2D NOESY experiment

A 
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 ~~~~~~~---
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28
Q

How do you work out the separation from NOESY

A

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

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29
Q

How do you use NOESY for sequential walking

A

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

30
Q

What is the definition of a short range NOE

A

less than 5 residues apart: used to detect alpha helix

31
Q

What is a long range NOE

A

more than 5 residues apart

32
Q

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

A

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

33
Q

What is the characteristic NOE of a beta sheet

A

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

34
Q

How do you distinguish an alpha helix from a 310 helix

A

No CaHi - NHi+2

35
Q

Define weak, medium and strong cross peaks

A

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

36
Q

What is simulated annealing

A

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

37
Q

What is the protocol of simulated annealing

A

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

38
Q

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

A
  • 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?
39
Q

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

A

Loops and termini: flexibility for binding and function

40
Q

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

A

coordinate root mean square displacement vs average structure

41
Q

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

A

rmsd vs “true” structure

42
Q

How do we reduce the complexity of NMR experiments

A

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

43
Q

How do we label proteins with 15N

A

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

44
Q

What is HSQC and why is it good

A

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

45
Q

How would you go about getting 13C proteins

A

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!!

46
Q

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

A

2D NOESY overwhelms space on the correlation map

47
Q

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

A

Can simplify the fingerprint region but not the aliphatic region

48
Q

What is the benefit of having 13C

A

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

49
Q

Describe stages in a triple resonance experiment

A

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

50
Q

Give the 5 stages of SAR by NMR for drug research

A

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)

51
Q

Why is SAR by NMR useful

A

Reduces discovery and synthesis time

Good for target directed synthesis

52
Q

What are the two mechanisms of spin relaxation

A
  • Longitudinal or spin-lattic relaxation (T1 time constant)

- Transverse or spin-spin relaxation (T2 time constant)

53
Q

What is longitudinal or spin-lattice relaxation

A

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

54
Q

What is transverse or spin-spin relaxation

A

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
~~~~~~~~~—

55
Q

What is the Fourier Transform

A

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

56
Q

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

A

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

57
Q

How does spin-echo for measuring T2 work

A

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

58
Q

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

A
  • 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
59
Q

What are the 3 mechanisms by which transverse relaxation occurs

A

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

60
Q

What is T1 affected by

A

fluctuations perpendicular to B0

61
Q

What is T2 affected by

A

fields along z

62
Q

How do we observe protein dynamics

A

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

63
Q

What is the timescale of overall motion of a protein

A

nanoseconds

64
Q

What is the timescale of motion of flexibility

A

picoseconds

65
Q

What is the problem with high molecular weight compounds

A

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

66
Q

Why do we use deuterium labelling for large molecules

A

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

67
Q

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

A

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

68
Q

What is TROSY

A

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

69
Q

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

A

Fast relaxation components appear broad

and slow relaxation components appear sharp

70
Q

Which component does the pulse sequence select

A

Just the fast component

Organic (4 decks)

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