Presentation on theme: "At this point, we have used COSY and TOCSY to connect spin"— Presentation transcript:
1At this point, we have used COSY and TOCSY to connect spin systems. i.e. if there are 8 arginines in the protein, we would aimto find 8 arginine patterns. Overlap or missing signals mayhamper 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.
2Consider 2 protons, I and S, not J-coupled but close in space W1 is the normal transition - gives rise to a peak in the spectrumbbW1IW1sW2 flip flipW0 flip flopbaabW1IW1SaaW1 requires frequencies or magnetic field fluctuations near theLarmor precession frequency i.e. 108 or 109 (100 MHz to 1000 MHz).W2 requires frequencies at wI + ws, or to a good approximation, 2wI.Wo requires frequencies at wI-ws, or to a good approximation.So what fields are present that can cause this relaxation?
3Spectral Density 2 - Rotational correlation time tc small molecules tumble more quicklylarge molecules tumble more slowlyrotational correlation time [in ns]approx. equal to 0.5 molecular mass [in kDa]1 kDa = 1000 atomic mass units
4For a small molecule, tc is small (~0 For a small molecule, tc is small (~0.3ns) and the product wtc is << 1In 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 frequenciesare required. Therefore this is the preferred mechanism in largemolecules. It is known as cross-relaxation.
5In the 2D NOESY experiment, an additional mixing time is added to the basic COSY sequence. The result is a build upof magnetisation from one nucleus to a close neighbour.90o90o90ot2t1Mixing time(magnetisation components of interest lie along -z)Cross relaxation now occurs to nearby nuclei.
6The 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
7The 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.