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Areas of Spectrum Remember - we are thinking of each amino acid as a spin system - isolated (in terms of 1H-1H J-coupling) from the adjacent amino acids by the peptide bond. H3C Yes 3J 2-10 Hz O C C CH H CH3 CH2 C C N H O 4 No J H O N C C Typical Amino Acid spin-system patterns on COSY spectra 1.) Just see 3J coupling 2.) Do not see couplings across the peptide bond. 3.6-4.5ppm CH3 1.4-1.7ppm Areas of Spectrum COSY Fingerprint region correlating NH-H protons COSY Spectrum of a small protein The ambiguity of a COSY spectrum Diagonal X’ X M,M’ A’ A A A’ M,M’ X X’ If we had some form of SUPER COSY that could go through many bonds at once we might get…. X’ X M,M’ A’ A 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 1H 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 90o t1 t2 Spin locking field The spin locking field (a series of rapid 90o pulses of varying phase) effectively averages the coupling 1H-1H 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. J13=0.2 Hz 1 2 3 J12=7 Hz J23=5 Hz Even if J13 is very small, will still see transfer to it via 2 8.83ppm 3.95ppm 1.52 bCH3 1.52ppm ALA 49 Ala49 3.95 H 1.52 bCH3 3.95 H 8.83ppm 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. 90o t1 90o 90o Mixing time t2 (magnetisation components of interest lie along -z) Cross relaxation now occurs to nearby nuclei. 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 1 NOE 6 r nuclear separation If we calibrate this function by measuring a known distance in the protein and the intensity of the NOE, we can write 1 NOE k 6 r where k is a proportionality constant 1 2 Sequential ‘walking’ with sequential nOes Fingerprint region dH of a 2D NOESY TOCSY gH bH 3 4 5 COSY 9.0 COSY TOCSY NOE 8.0 O C CH3 N C C H H O NOE COO CH2 H H O N C C CH2 Ala CH H3C - CH3 N H H 7.0 NH NH-NH Contacts NOE O C CH3 N C C H H O 1 COO H H O N C C CH2 Ala CH H3C dH - CH3 CH2 N H gH 2 bH 3 H 4 5 9.0 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. 8.0 7.0 Connectivites by NOE dN - Connects CH of residue i to NH of i+1 dbN - Connects CbH of resdiue i to NH of i+1 dNN - Connects NH of residue i to NH of i+1 LEU dN H dN dNN N dbN H ALA VAL C dNN C H3C H N CH3 CH d O bN C C H N C H CH3 dNN O H dN CH2 dbN Hi-NHi+3 Hi-NHi+1 i+3 H i+2 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 N i+4 the sequence (sequential nOes H connect neighbouring residues) NOE H For an -helix we see Hi-NHi+3 and Hi-NHi+4 nOes. i A b-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