Modern approaches to protein Flashcards
What does the gyromagnetic ratio determine
The gyromagnetic ratio determines the ratio of nuclear magnetic moment to nuclear spin
What do fundamental symmetry theorems predict
Fundamental symmetry theorems predict co-linear spin and magnetic moment
What is the equation for magnetic moment
u=gamma x I
How much magnetism (u) we get for spin I
What does energy depend on
size and relative orientation of B and u
What is Zeeman splitting
delta E = gamma hbar B0
split by different values of m
What is the Larmor frequency
Resonance corresponding to the Zeeman splitting induced in NMR experiments
w0 = gamma B0
Why do we need a large magnetic field
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
Give the protocol of a 1D FT NMR experiment
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
Why do we run experiments in D2O
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
How do we use pH to monitor a peak for example which protons are bound to an active site
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
What are the 3 stages to an NMR experiment
resolve resonances, assign, interpret data
What is COSY
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
Typically whats the J limit you can see in COSY
3J: you do not see across the peptide bond
What is a spin system
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
What do you see in the 0-5 ppm region of a COSY
amino acid side chain spin systems sitting on top of each other
What is the fingerprint region of a COSY
Calpha - NH region;
top axis: 6.5 - 9 ppm
sideaxis: 3.5 - 5 ppm
Where do you see Aromatic-aliphatic peaks in a COSY
top axis: 6.5 - 8.5 ppm
side axis: 0 - 2.5 ppm
Why can ambiguity arise from a COSY spectrum
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
What is TOCSY
Total correlation spectroscopy: produces cross peaks between all protons of a spin system and is therefore ideal for proteins
What is a spin locking field
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
How come we can correlate along the entire system in TOCSY
Dispersion of the NH-aH region
What does NOESY allow
It provides data on internuclear distances and therefore you can correlate directly with molecular structure
What is an nOe caused by
Cross relaxation: think of that energy level diagram with aa bb ab ba W2 is aa bb and W0 is ab ba
In small molecules which is the dominant NOE effect
W2 because of rapid tumbling
In large molecules which is the dominant NOE effect
W0: negative NOE enhancement
W0 connects energy levels of similar energy so only low frequency required
“cross relaxation”
How do you carry out a 2D NOESY experiment
add additional mixing time to the COSY experiment and see a build up of magnetisation from one nucleus to a close neighbour
Describe the protocol of a 2D NOESY experiment
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 ~~~~~~~---
How do you work out the separation from NOESY
NOE = k (1/r6) so calibrate with known distance where k is a proportionality constant
Detect up to 5A
How do you use NOESY for sequential walking
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
What is the definition of a short range NOE
less than 5 residues apart: used to detect alpha helix
What is a long range NOE
more than 5 residues apart
What is the characteristic NOE of an alpha helix (3 strong, 1 weak)
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
What is the characteristic NOE of a beta sheet
aHi - NHi+1 and long range NOE. This is always very strong
How do you distinguish an alpha helix from a 310 helix
No CaHi - NHi+2
Define weak, medium and strong cross peaks
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
What is simulated annealing
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
What is the protocol of simulated annealing
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
What are the challenges for interpreting 3D structures (2)
- 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?
In which parts of a molecular structure do you see poor definition in NMR experiments
Loops and termini: flexibility for binding and function
How do we work out the precision of an NMR determined structure
coordinate root mean square displacement vs average structure
How do we work out the accuracy of an NMR determined structure
rmsd vs “true” structure
How do we reduce the complexity of NMR experiments
use 15N as it is a spin 1/2 nucleus
How do we label proteins with 15N
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
What is HSQC and why is it good
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
How would you go about getting 13C proteins
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!!
Why do you need triple resonance experiments (why is NOESY not enough)
2D NOESY overwhelms space on the correlation map
Which region of the 2D map can 15N simplify and which region can it not simplify
Can simplify the fingerprint region but not the aliphatic region
What is the benefit of having 13C
Spin active 13C=O allows correlation between amino acids so no longer acts as a brick wall
Describe stages in a triple resonance experiment
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
Give the 5 stages of SAR by NMR for drug research
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)
Why is SAR by NMR useful
Reduces discovery and synthesis time
Good for target directed synthesis
What are the two mechanisms of spin relaxation
- Longitudinal or spin-lattic relaxation (T1 time constant)
- Transverse or spin-spin relaxation (T2 time constant)
What is longitudinal or spin-lattice relaxation
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
What is transverse or spin-spin relaxation
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
~~~~~~~~~—
What is the Fourier Transform
The fourier transform is a computational method for analysing frequencies in an oscillating signal
Why doesn’t a normal experiment to measure T2 work well
Poor shimming and field inhomogeneity: field dephases due to inhomogeneity during tau/2
How does spin-echo for measuring T2 work
Each magnetisation vector is rotated about y by a second pulse then they reconverge = echo
What are the two mechanisms by which a system returns to equilibrium through spin-lattice relaxation
- 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
What are the 3 mechanisms by which transverse relaxation occurs
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
What is T1 affected by
fluctuations perpendicular to B0
What is T2 affected by
fields along z
How do we observe protein dynamics
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
What is the timescale of overall motion of a protein
nanoseconds
What is the timescale of motion of flexibility
picoseconds
What is the problem with high molecular weight compounds
slower tumbling leads to fast decay which gives poor signal to noise
Why do we use deuterium labelling for large molecules
For molecules above 25kDa 2D labelling reduces the relaxation (gD/gH)~1/0.5 which improves the signal to noise ratio for better resolution
Which n-bonded protons do you see in a deuterium labelled experiment
Just the non-labile N-1H so on 15N you just see N-1H protons which makes a better spectrum
What is TROSY
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
What components are there to relaxation, how do they appear in a spectrum
Fast relaxation components appear broad
and slow relaxation components appear sharp
Which component does the pulse sequence select
Just the fast component
