10. Lecture 9 Notes PDF

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Lecture Notes...

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Through-bond J-coupling and 2D, 3D NMR Learning objectives: 1. 2. 3. 4. 5. 6. 7.

Recognise J coupling, connects nuclei that are linked together through bonds (up to 4 bonds) Explain that the splitting pattern will tell us how many neighbouring nuclei are present Describe the relationship between the backbone torsion angles in proteins and the 3Ja-NH coupling illustrate how 2D NMR can be used to connect NMR peaks that are connected through bonds Describe how amino acids in proteins have standard patterns of cross-peaks in 2D spectra Report why a 2D 15N HSQC gives a “fingerprint” of a protein Explain how 3D NMR experiments separate out crowded 2D NMR spectra

Information available from 1D 1H NMR spectra 1. Chemical shif  Gives information on the chemical environment of a particular H 2. Integration  Area under curve  Number of protons are equivalent to the peak 3. Multiplicity  Splitting= neighbouring H  N+1 peak rule 4. Coupling constant Spin-spin (through-bond) J-coupling 

Principle: - Through-bond J-coupling is due to the coupling/linking of neighbouring nuclei - The possible spin orientation (with or against the magnetic field) of neighbouring nuclei effects the magnetic field of the nucleus being studied via the electron distribution around the nucleus. - The nuclei must be linked/coupled via their electron in covalent bond - Both north pole are up spin - If the 2nd north pole of magnets flips to the south pole it will experience a different magnetic field - The 2st H nuclei can experience 2nd H nuclei in 2 possible ways (aligned or against applied field), which the nmr can detect the difference - Due to this, get peaks at a slightly different frequency as experiencing a slightly different magnetic field and therefore the nucleus will wobble at a different frequency

Splitting patterns       

Gives information about neighbouring H The resonant frequencies increase depending on if the neighbouring nuclei. This produces a splitting patters In proton NMR a peak with singlet indicates no through-bond neighbours. A doublet indicates 1 through bond neighbour A triplet indicated 2 neighbours A quartet three neighbour H

Appearance of some multiplet   

The magnitude of the coupling give information and its called J coupling Magnitude of J coupling measured in Hz rather than ppm so it’s a radio wave frequency J-values are independent of the magnetic field strength

J coupling

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Coupling is transmitted through chemical bonds J coupling occurs between 2,3 or 4 bonds apart

Magnitude of J values 

Effect by number, type and angle of bonds

Torsion angles from the magnitude of coupling constants  The size of the coupling J is measured in Hz and is influenced by the nature of the bond between the coupled nuclei  3Jalpha N coupling in the protein backbone: - The amind H has 1 through bond H neighbours H alpha - The amind H is therefore a doublet - The torsion angle between the Ca-H and N-H bond will affect the size of the coupling constant

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The relationship is described by the karplus curve Sigmoidal relationship called karplus curve

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Y axis = magnitude of coupling

2D NMR Spectra-COSY & TOCSY 

2D Experiments: - COSY= correlation spectroscopy - TOCSY= total correlation spectroscopy - 2D TOCSY & COSY connect H that are linked by a bond (spin-spin coupled) - Spectra normally shown as countour map - Diagonal peak are same as 1D each proton is a peak - Off diagonal peak (cross peaks) link individual H - Not close in space but linked by 2 or 3 covalent bonds

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2D spectrum i.e. TOCSY 55 aa protein domain Approx.. 1000 cross peaks

1H NMR Assignment of protein resonance’s   

Peaks in the side chain of aa give a characteristics spin system pattern Different aa have different pattern of linking 20 aa so 20 spin patterns

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Solid line represents COSY experiment Dotted line represents TOCSY experiment

For larger proteins- correlating 1H with 14-N and 13C

3D NMR experiment using 15N and 13C    

As the molecule understudy gets bigger so do the number of H and therefore the number of NMR peaks Larger than 70 aa 2D becomes too crowded and 3D are required 3D comes from hetro-nuclei: 15N and 13 C Hetero-nuclear coupling constants 15N and 13C is also used

15N HSQC for prion protein ‘finger print’ spectra   

120 aa 120 cross-peaks One for each NH proton on the backbone

NMR experiment with 3 coupled nuclei 1H,15N and 13C

Summary        

Spin-spin J coupling, connects nuclei that are linked together through bonds (up to 4 bonds) The splitting pattern will tell us how many neighbouring nuclei there are The size of the coupling can tell us the torsion angles between nuclei The backbone torsion angles in proteins can be measured via the 3Ja-NH coupling 2D NMR can be used to connect peaks that are coupled through bonds Amino acids in proteins have standard patterns of cross-peaks in 2D spectra 2D 15N HSQC gives a cross peak for each amide in the backbone 3D NMR experiments separate out crowded 2D spectra into a third dimension using 15N or 13C...