Areas of Spectrum

Remember - we are thinking of each amino acid as a spin system - isolated (in terms of 1 H- 1 H J-coupling) from the adjacent amino acids by the peptide bond.

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

CH ppm ppm

Areas of Spectrum

COSY Fingerprint region correlating NH-  H protons

COSY Spectrum of a small protein

The ambiguity of a COSY spectrum AA’M,M’XX’ A A’ M,M’ X X’ Diagonal

If we had some form of SUPER COSY that could go through many bonds at once we might get…. AA’M,M’XX’ A A’ M,M’ X X’ Diagonal

TOCSY - Total correlation Spectroscopy To relieve overlap and ambiguity, methods developed to overcome them. One popular method is TOCSY. Basic aim is to produce cross peaks between all of the 1 H spins which belong to the same spin system Ideal for proteins where each amino acid is a self contained spin system, separated by the peptide bond. To understand (quickly!) what TOCSY is we need to introduce the concept of a spinlock.

TOCSY t2t2 90 o t1t1 Spin locking field The spin locking field (a series of rapid 90 o pulses of varying phase) effectively averages the coupling 1 H- 1 H coupling constants over the entire spin system. The dispersion of the NH-  H region allows correlations along the entire system to become visible.

Homonuclear Hartmann-Hahn and TOCSY experiments Under these conditions magnetisation is transferred very efficiently, at a rate determined by J, between coupled nuclei. The longer the mixing time, the further through the spin system the magnetisation propagates J 12 =7 HzJ 23 =5 Hz J 13 =0.2 Hz Even if J 13 is very small, will still see transfer to it via 2

3.95ppm 1.52ppm Ala  H 1.52  CH 3 ALA  H 1.52  CH ppm

t2t2 90 o t1t1 (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.

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 the protein and the intensity of the NOE, we can write where k is a proportionality constant

NH Fingerprint region of a 2D NOESY Ala COSY TOCSY HH HH HH NOE TOCSY COSY Sequential ‘walking’ with sequential nOes

NH-NH Contacts Ala HH HH HH NOE The ‘NH-NH’ region provides an additional source of sequential contacts - note the symmetry around the diagonal and that this contact does not give direction.

Connectivites by NOE LEUVAL ALA

 Hi-NHi+1  Hi-NHi+3

H i i+3 H i+2 NOE H N An  -helix can be recognised by repeating patterns of short range nOes. A short range nOe is defined as a contact between residues less than five apart in the sequence (sequential nOes connect neighbouring residues) For an  -helix we see  H i -NH i+3 and  H i -NH i+4 nOes. i+4

A  -strand is distinguished by strong C  H i -NH i+1 contacts 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