Areas of Spectrum

Examples of aliphatic region correlations Ala

Thr

3.95

1.52

Asp

Areas of Spectrum

The fingerprint region – the 2nd region of interest in the COSY spectrum

COSY Fingerprint region correlating NH-H protons

COSY Spectrum of a small protein

Fingerprint region

Aliphatic

Total correlation spectroscopy - TOCSY

t290o

t1

Spin locking field

The spin locking field (a series of rapid 90o pulses ofvarying phase) effectively averages the coupling 1H-1Hcoupling constants over the entire spin system.

The dispersion of the NH-H region allows correlations alongthe entire system to become visible.

Water Presaturation

Homonuclear Hartmann-Hahn or TOCSY experiments

Under these conditions magnetisation is transferred very efficiently,at a rate determined by J, between coupled nuclei. The longer themixing time, the further through the spin system the magnetisationpropagates.

1 2 3

J12=7 Hz J23=5 Hz

J13=0.2 Hz

Even if J13 is very small, will still see transfer to it via 2

3.95ppm

1.52ppm

Ala49 3.95 H 1.52 CH3

ALA 49

3.95 H

1.52 CH3

8.83ppm

8.83ppm

At this point, we have used COSY and TOCSY to connect spin systems. i.e. if there are 8 arginines in the protein, we would aim to find 8 arginine patterns. Overlap or missing signals may hamper us in this initial goal. The next step is to use NOESY experiments to

sequentially link the amino acid spin systems together.

The nuclear Overhauser enhancement provides data on internuclear distances. These can be more directly correlated with molecular structure.

Connecting spin systems – The nuclear Overhauser effect (nOe)

W2 flip flip

W0 flip flop

Consider 2 protons, I and S, not J-coupled but close in space

W1s

W1I W1S

W1I

W1 is the normal transition probability that gives rise to a peak in the spectrum

W1 requires frequencies or magnetic field fluctuations near the Larmor precession frequency i.e. (e.g. 500 MHz at 11.1 Tesla).W2 requires frequencies at wI + ws, or to a good approximation, 2wI or 109 Hz Wo is a zero quantum transition that requires frequencies at wI-ws, i.e. just the chemical shift difference of the protons which could be 0 to a few 1000 Hz)

Rotational correlation time c

rotational correlation time [in ns] is approx. equal to 0.5 molecular mass [in kDa]

A transition corresponding to a given frequency is promoted by molecular motion at the same frequency. Small molecules in non-viscous solvents tumble at rates around 1011 Hz, while larger molecules such as proteins tumble at rates around 107 Hz. For small molecules, W2 will be greater than W0 and this is the dominant mechanism for producing NOE enhancements (which turn out be positive)For larger molecules W0 will become greater than W2 and this becomes the dominantmechanism leading to NOE enhancements (that are now negative).

In the energy level diagram for a 2 spin system, it is the transitions that involve a simultaneous flip of both spins (cross - relaxation) that cause NOE enhancements.

For a small molecule, c is small (~0.3ns) and the product c is << 1. In this extreme narrowing limit, rotational motions include 2o (i.e. fast motions) and W2 is preferred.

In large molecules (PROTEINS!), the tumbling is slow and c > 1. Wo connects energy levels of similar energy so only low frequencies are required. Therefore this is the preferred mechanism in large molecules. It is known as cross-relaxation.

t2

90o90o

t1

90o

(magnetisation components of interest lie along –z). Cross relaxation now occurs to nearby nuclei.

Mixing time

In the 2D NOESY experiment, an additional mixing time is added to the basic COSY sequence. The result is a build up of magnetisation from one nucleus to a close neighbour.

Presat

The NOE operates ‘through space’, it does not require the nuclei to be chemically bonded. The build-up is proportional to the separation of the two nuclei

nuclear separation

If we calibrate this function by measuring a known distance in theprotein and the intensity of the NOE, we can write

where k is aproportionalityconstant

The power of the NOESY experiment is that the intensity of an NOE peak will be related to the nuclear separation.

Strong NOE crosspeaks - 2.5 ÅWeak NOE crosspeaks - 2.5-3.5 ÅExtending the mixing time will permit nuclei separated by 5Å - notall spin systems will give a detectable peak though. So the absence ofa peak does not preclude close approach. Similarly a weaker crosspeak does not always prove a larger internuclear distance.

Therefore tend to be cautious and define distance ranges.Strong (1.8-2.5Å), medium (1.8-3.5Å), weak (1.8-5.0Å).

Since this works through space we can use the NOE to connect spinsystems that we assigned with the COSY and TOCSY spectra.

NH7.09.0 8.0

1

2

3

4

5

Fingerprint regionof a 2D NOESY

Ala

COSY

TOCSY

H

H

H

NO

E

NOE

NOE

TOCSY

COSY

Sequential ‘walking’ with sequential nOes

NH-NH Contacts

7.09.0 8.0

1

2

3

4

5

Ala

H

H

H

NOE

The ‘NH-NH’ region provides an additional source of sequential contacts - note the symmetryaround the diagonal and thatthis contact does not give direction.

Hi-NHi+1Hi-NHi+3

H

i

i+3

Hi+2

NOE

HN

An -helix can be recognisedby repeating patterns of shortrange nOes. A short range nOeis defined as a contact betweenresidues less than five apart inthe sequence (sequential nOesconnect neighbouring residues)

For an -helix we see Hi-NHi+3

and Hi-NHi+4 nOes.

i+4

A -strand is distinguished by strong CHi-NHi+1contacts and long range nOes connecting the strands.

A long range nOe connects residues more than 5 residues apart in the chain.

Assignment of secondary structural segments

• sequential stretches of residues with consistent secondary structure characteristics provide a reliable indication of the location of these structural segments