2 Examples of aliphatic region correlations Ala1.523.95ThrAsp
3 The fingerprint region – the 2nd region of interest in the COSY spectrum Areas of Spectrum
4 COSY Fingerprint region correlating NH-aH protons
5 COSY Spectrum of a small protein AliphaticFingerprint region
6 Total correlation spectroscopy - TOCSY Water PresaturationSpin locking fieldThe 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-aH region allows correlations alongthe entire system to become visible.
7 Even if J13 is very small, will still see transfer to it via 2 Homonuclear Hartmann-Hahn or TOCSY experimentsUnder 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.J13=0.2 Hz123J12=7 HzJ23=5 HzEven if J13 is very small, will still see transfer to it via 2
9 Connecting spin systems – The nuclear Overhauser effect (nOe) 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.
10 W1I W1s W2 flip flip W0 flip flop W1I W1S Consider 2 protons, I and S, not J-coupled but close in spaceW1 is the normal transition probability that gives rise to a peak in the spectrumbbW1IW1sW2 flip flipW0 flip flopbaabW1IW1SaaW1 requires frequencies or magnetic field fluctuations near theLarmor 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 HzWo is a zero quantum transition that requires frequencies at wI-ws, i.e. just thechemical shift difference of the protons which could be 0 to a few 1000 Hz)
11 Rotational correlation time tc In the energy level diagram for a 2 spin system, it is the transitions that involvea simultaneous flip of both spins (cross - relaxation) that cause NOEenhancements.A transition corresponding to a given frequency is promoted by molecular motionat the same frequency. Small molecules in non-viscous solvents tumble at ratesaround 1011 Hz, while larger molecules such as proteins tumble at rates around107 Hz. For small molecules, W2 will be greater than W0 and this is the dominantmechanism 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).Rotational correlation time tcrotational correlation time [in ns] is approx. equal to 0.5 molecular mass [in kDa]
12 For a small molecule, tc is small (~0 For a small molecule, tc is small (~0.3ns) and the product wtc is << 1. In this extreme narrowing limit, rotational motions include 2wo (i.e. fast motions) and W2 is preferred.In large molecules (PROTEINS!), the tumbling is slow and wtc > 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.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.90o90o90ot2t1Mixing timePresat(magnetisation components of interest lie along –z). Cross relaxation now occurs to nearby nuclei.
13 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 nucleinuclear separationIf we calibrate this function by measuring a known distance in theprotein and the intensity of the NOE, we can writewhere k is aproportionalityconstant
14 The power of the NOESY experiment is that the intensity of an NOE peak will be related to the nuclear separation.Strong NOE crosspeaks ÅWeak NOE crosspeaks 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 weakercrosspeak 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 spinsystems that we assigned with the COSY and TOCSY spectra.
15 Sequential ‘walking’ with sequential nOes 12345Fingerprint regionof a 2D NOESYdHTOCSYgHbHCOSYNH9.08.07.0NOECOSYNOETOCSYNOEAla
16 NH-NH Contacts dH NOE gH bH Ala 9.0 8.0 7.0 12345dHNOEgHbHAla9.08.07.0The ‘NH-NH’ region provides anadditional source of sequentialcontacts - note the symmetryaround the diagonal and thatthis contact does not give direction.
18 An a-helix can be recognised by repeating patterns of short range nOes. A short range nOeis defined as a contact betweenresidues less than five apart inthe sequence (sequential nOesconnect neighbouring residues)For an a-helix we see aHi-NHi+3and aHi-NHi+4 nOes.i+4i+3NHHNOEHi+2i
19 A b-strand is distinguished by strong CaHi-NHi+1contacts and long range nOes connecting the strands. A long range nOe connects residues more than 5 residues apart in the chain.
20 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