A one-dimensional (1D) NMR spectrum of a protein HNHN HH Chemical shifts in parts per million (ppm) Are independent of the field strength of the Static magnetic Bo field. See the supplementary lecture material and Rattle, ‘NMR Primer for Life Scientists Pages 19-21, Backbone H N Aromatics H2OH2O HH Upfield shifted methyls Methyl H chemical shift (ppm) For a 1 H protein spectrum we need a fair amount of protein, maybe 5-10 mgs of protein. 600  L sample volume - gives a 1-2 mM concentration of protein.

The 1D 1 H spectrum of a protein ppm Amides Aromatics H2OH2O CHCH CHCH C  H C  H Upfield shifted methyl

A lot of work done with histidine since the C2 proton appears at higher frequency than most other protons ppm ppm

1 H NMR spectrum of Histidine - C2 proton appears at higher frequency than most other protons and is sensitive to the protonation of the ring. C2HC4H CHCH CHCH 0 10 Raise pH 10 0ppm ppm ppm Shown in protonated form  

pH Chemical Shift Change  ppm) 0 1 Titration of the C2H of Histidine pKa = 5.2 Shift measured with multiple 1D spectra starting with pH 1.0 and moving through to pH 9 The chemical shift change of the proton on C2 reflects the protonation of N1 50% of complete change

4 histidines which could be monitored and have their pKa’s measured.

H1 = His105 H2 = His119 H3 = His12 H4 = His48 Measure pK a of each histidine pKa His His His His48 is more complex, sudden discontinuity in the curve.

Found that 200mM Na + CH 3 COO - helped to stabilize the protein. Can then determine that the pKa of C2H is There is a conformational change affecting this peak so that at some pHs two peaks were observed. H4a and H4b were acid and base stable forms.

His105 His12 His48 His119 His48 and His105 are unchanged His12 and His119 curved are shifted downfield. Why downfield?? His119 changes from 6.2 to 8.0 His 12 changes from 5.8 to 7.4 Both His12 and His119 are protonated in the enzyme- inhibitor complex. The proton is protected from exchange by the presence of the inhibitor. Need to go to higher pH to remove it. Repeat titrations in the presence of an inhibitor. in this case, cytidine-3’- monophosphate (3’-CMP)

A simple 1D NMR Spectrum - this is ethanol but the spectrum has the sort of simplicity we might get with a one amino acid protein (there is no such thing!) A 36 amino acid protein A successful NMR experiment comes in 3 stages, 1.) Resolve the resonances - that is, obtain a spectrum where individual signals are clearly resolved from one another. 2.) Assign the resonances. Each peak comes from one atom in the protein - but which one? Our 36 amino acid protein is a mess! The record to date is 723 amino acids With full assignment of the spectrum - how did they do this? 3.) Interpret the data.

Effect of increasing spectrometer frequency 1 GHz soon??

13 rad s -1 rad s -1 T -1. T s -1 (Hz) Larmor Frequency

14 A compass in a magnetic field

15 A nuclear spin precesses in a magnetic field the circulating motion of the spin angular momentum is called precession Nuclear spins precess because: they are magnetic they have angular momentum this arrow denotes the direction of the spin angular momentum

16 Precession frequency = Larmor frequency 0 = -  B o /2π Larmor frequency in Hz (= cycles per second) gyromagnetic ratio in rad s –1 T –1 magnetic field in Tesla (T) Compare with Zeeman Splitting

x y z BoBo   Magnetic vectors precess around the static magnetic field at the larmor frequency. There is a slight population excess in the low energy (  ) state. This leads to a net magnetization along Z (in green). There is no net magnetisation along x or y as this is essentially randomised. x y   x y  

Generation of transverse magnetization by π/2 pulse x y z (  /2) x x y z  /2 Magnetization perpendicular to the magnetic field is called transverse magnetization y Net Moment

NMR signal Note the orientation of the coil - perpendicular to the magnetic field x y z BoBo Preamp The NMR signal is also called the free induction decay (fid)

Precession of Transverse Magnetization The transverse magnetization components oscillate and decay MxMx MyMy Time x y z x y z x y z BoBo xy plane M y (t) = -M z eq cos(   t) exp{-t / T 2 } M x (t) = M z eq sin(   t) exp{-t / T 2 } oscillation at the Larmor frequency decay time constant = spin-spin relaxation time OR transverse relaxation time

Pg 46 & 47 of Rattle

Diagonal Crosspeaks

COSY of Alanine in D 2 O

COSY Spectrum of a small protein

Areas of Spectrum

Typical Amino Acid spin-system patterns on COSY spectra 1.) Just see 3 J coupling 2.) Do not see couplings across the peptide bond.