1 / 23

Biomolecular Nuclear Magnetic Resonance Spectroscopy

01/28/08. Biomolecular Nuclear Magnetic Resonance Spectroscopy. BASIC CONCEPTS OF NMR How does NMR work? Resonance assignment Structural parameters. Reading: Chapter 22 in Protein and Peptide Drug Analysis “Solution Structure Determination of Proteins by NMR”. Nuclear Spin.

badru
Download Presentation

Biomolecular Nuclear Magnetic Resonance Spectroscopy

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 01/28/08 Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR • How does NMR work? • Resonance assignment • Structural parameters Reading: Chapter 22 in Protein and Peptide Drug Analysis “Solution Structure Determination of Proteins by NMR”

  2. Nuclear Spin • Nuclear spin angular momentum is a quantized property of the nucleus in each atom, which arises from the sub-atomic properties of neutrons and protons • The nuclear spin angular momentum of each atom is represented by a nuclear spin quantum number (I) • All nuclei with even mass numbers have I=0,1,2… • All nuclei with odd mass numbers have I=1/2,3/2... • NMR is possible with all nuclei except I=0, but I=1/2 has simplest physics Biomolecular NMR  primarily1H, 13C, 15N (31P)

  3. Efficiency factor- nucleus DE = h g Bo Constants Strength of magnet Spin 1/2 Nuclei in a Magnetic Field Bo Energy

  4. Intrinsic Sensitivity of Nuclei Nucleusg % Natural Relative Abundance Sensitivity 1H 2.7 x 108 99.98 1.0 13C 6.7 x 107 1.11 0.004 15N -2.7 x 107 0.36 0.0004 31P 1.1 x 108 100 0.5 Prepare samples enriched in these nuclei

  5. Sensitivity (S) ~ D pop. (N vs N) Efficiency factor- nucleus N N -DE/kT = e S ~ DN = DE = h g Ho Constants Strength of magnet Variables Affecting Sensitivity - DE is very small DN is small - DN ~ 1:105 (at room T) NMR has low sensitivity  requires lots of sample! Increase sensitivity by increasing magnetic field strength or reducing electronic noise (cryo-probes)

  6. Bo DE Equilibrium B1 Pump in energy (RF transmitter) hn = DE Non-equilibrium NMR signals Release energy (RF receiver) hn = DE Equilibrium The Resonance Experiment Strength of signal  D (population)

  7. NMR TerminologyChemical Shift & Linewidth The exact resonance frequency (chemical shift) is determined by the electronic environment of the nucleus

  8. Scalar and Dipolar Coupling Through Space Through Bonds • Coupling of nuclei gives information on structure

  9. OH CH2 CH3 The key attribute: use the scalar and dipolar couplings to match the set of signals with the molecular structure Resonance Assignment CH3-CH2-OH Which signal from which H atoms?

  10. F1 F2 2D NMR Spectra FacilitateIdentification of Coupling Coupled spins

  11. Biomolecules Have Many Signals 1H NMR Spectrum of Ubiquitin ~75 residues, ~500 1H resonances • Terminology: signals are overlapped

  12. Challenges For Using NMR to Study Biological Macromolecules • Hundreds-thousands of signals! • Must assign the specific signal for each atom • Thousands of couplings between nuclei- these also need to be assigned

  13. Critical Features of Protein NMR Spectra • Regions of the spectrum correspond to different parts of the amino acid • Tertiary structure leads to increased dispersion of resonances

  14. Regions of the 1H NMR Spectrumand Dispersion by the 3D Fold What would the unfolded protein look like?

  15. Critical Features of NMR Spectra of Biomolecules • Regions of the spectrum correspond to different parts of the amino acid • Tertiary structure leads to increased dispersion of resonances • Bio-macromolecules are polymers  The nuclei are coupled to some (but not all!) other nuclei

  16. Spectra of Biomacromolecules:Overlapped Sub-Spectra *Groups of coupled nuclei* Each residue in the sequence gives rise to an independent NMR sub-spectrum, which is much simpler than the complete spectrum Methods have been developed to extract each sub-spectrum from the whole

  17. T L R S G G S Strategy to AssignResonances in a Protein • Identify resonances for each residue (scalar) • Put amino acids in order (dipolar) 1 2 3 4 5 6 7 R - G - S - T - L - G - S Same idea for any biopolymer (e.g. DNA, RNA)

  18. Even Sub-Spectra are Overlapped! 1H NMR Spectrum of Ubiquitin • Resolve resonances by multi-dimensional experiments

  19. Use 2D NMR to Resolve Overlapping Signals Sub-spectra overlapped 1D Crosspeaks resolved! 2D Coupled spins

  20. Hb Ha HN Multi-Dimensional NMR If 2D cross peaks overlap go to 3D or 4D …..

  21. t2 t1 t3 Another Solution tothe Overlap Challenge • Increase dimensionality of spectra to better resolve signals: 1D2D3D4D…. • Use attached nuclei (13C,15N) to distinguish Hz HA

  22. Multi-Dimensional Heteronuclear NMR 15N-1H HSQC F1 Chemical Shift (15N) F2 Chemical Shift (1H)

  23. Advantages ofHeteronuclear nD NMR Uses a second nucleus to resolve overlap of the first: chemical shift of each nucleus is characteristic/sensitive to different factors More information to identify resonances Less sensitive to MW because this strategy uses large 1 and 2-bond scalar couplings

More Related