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Areas of Spectrum

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Examples of aliphatic region correlations Ala Thr Asp

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

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COSY Fingerprint region correlating NH- H protons

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COSY Spectrum of a small protein Fingerprint region Aliphatic

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Total correlation spectroscopy - 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. Water Presaturation

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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 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

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

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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)

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W 2 flip flip W 0 flip flop Consider 2 protons, I and S, not J-coupled but close in space W 1s W 1I W 1S W 1I W 1 is the normal transition probability that gives rise to a peak in the spectrum W 1 requires frequencies or magnetic field fluctuations near the Larmor precession frequency i.e. (e.g. 500 MHz at 11.1 Tesla). W 2 requires frequencies at w I + w s, or to a good approximation, 2w I or 10 9 Hz W o is a zero quantum transition that requires frequencies at w I -w s, i.e. just the chemical shift difference of the protons which could be 0 to a few 1000 Hz)

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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 Hz, while larger molecules such as proteins tumble at rates around 10 7 Hz. For small molecules, W 2 will be greater than W 0 and this is the dominant mechanism for producing NOE enhancements (which turn out be positive) For larger molecules W 0 will become greater than W 2 and this becomes the dominant mechanism 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.

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For a small molecule, c is small (~0.3ns) and the product c is << 1. In this extreme narrowing limit, rotational motions include 2 o (i.e. fast motions) and W 2 is preferred. In large molecules (PROTEINS!), the tumbling is slow and c > 1. W o 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. 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. Presat

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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

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The power of the NOESY experiment is that the intensity of an NOE peak will be related to the nuclear separation. Strong NOE crosspeaks Å Weak NOE crosspeaks Å Extending the mixing time will permit nuclei separated by 5Å - not all spin systems will give a detectable peak though. So the absence of a 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 ( Å), medium ( Å), weak ( Å). Since this works through space we can use the NOE to connect spin systems that we assigned with the COSY and TOCSY spectra.

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NH Fingerprint region of a 2D NOESY Ala COSY TOCSY H H H NOE TOCSY COSY Sequential walking with sequential nOes

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NH-NH Contacts Ala 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.

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Hi-NHi+1 Hi-NHi+3

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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

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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.

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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

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