Biomolecular Nuclear Magnetic Resonance Spectroscopy - PowerPoint - PowerPoint by HC120218094950

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									                                       01/23/04


Biomolecular Nuclear Magnetic
   Resonance Spectroscopy
 RESONANCE ASSIGNMENT IN PROTEINS
 • Multi-dimensional NMR experiments
 • NMR analysis of proteins
Proteins Have Too Many Signals!
       1H   NMR Spectrum of Ubiquitin

         ~500 resonances




Resolve resonances by multi-dimensional experiments
 The Pulse FT NMR Experiment
                                            90º pulse
Experiment
                                            (t)

             equilibration                  detection of signals


                                 Fourier
  Data                          Transform
 Analysis

              Time domain (t)
The 2D NMR Pulse Sequence

                  +
       1D + 1D = 2D




        [2nd preparation]
2D NMR: Coupling is the Key
             2D detect signals twice
             (before/after coupling)
 90º pulse


                                       t1        t2


                   Transfers between
Same as 1D           coupled spins          t1        t2
experiment
         The 2D NMR Spectrum

Pulse Sequence



                                t1     t2

  Spectrum

  Before mixing        Coupled spins



    After mixing
   The Power of 2D NMR:
Resolving Overlapping Signals
 1D                    2 signals
                      overlapped



 2D                  2 cross peaks
                        resolved
Acronyms For Basic Experiments
 Differ Only By The Nature Of Mixing
                   Homonuclear   Heteronuclear
Scalar Coupling      COSY           HSQC
                     TOCSY       Hetero-TOCSY
                    Multiple        HMQC
                    Quantum

Dipolar Coupling     NOESY       NOESY-HSQC
                                 NOESY-HMQC
      Higher Dimensional NMR:
       Built on the 2D Principle
                       3D- detect signals 3 times

      90º pulse

                                                          (t3)




                                                         t2
                                                    t1
     Same as 1D
     experiment                                               t3

                  3D NMR Pulse Sequence

Experiments are composites  acronyms are composites
Proteins Have Too Many Signals!
    1H   NMR Spectrum of Ubiquitin

    ~500 resonances
  Challenges For Determining
 Protein Structures Using NMR
• Proteins have thousands of signals
• Assign the specific signal for each atom
• Thousands of interactions between
  atoms- also need to be assigned
• Need to transform representation from
  spectrum through interactions between
  atoms to spatial coordinates
NMR Spectrum to 3D Structure

        H         H           H

        H     H           H
                      H

            H H H         H
        Critical Features of
       Protein NMR Spectra
•The nuclei are not all mutually coupled
    Each amino acid gives rise to an
independent NMR sub-spectrum, which is
 much simpler than the complete protein
               spectrum
        Critical Features of
       Protein NMR Spectra
• The nuclei are not all mutually coupled
• Regions of the spectrum correspond to
  different parts of the amino acid
• Tertiary structure leads to increased
  dispersion of resonances
Regions of the       1H   NMR Spectrum




    What would the unfolded protein look like?
    Solutions to the Challenges
1. Increase dimensionality of spectra to
   better resolve signals: 123 4

                         t2
                   t1


                              t3
  Resolve Peaks By Multi-D NMR
A BONUSregions in
2D spectra provide
protein fingerprints




If 2D cross peaks
overlap go to 3D
    Solutions to the Challenges
1. Increase dimensionality of spectra to
   better resolve signals: 123 4
2. Detect signals from heteronuclei
   (13C,15N)
    Better resolution of signals/chemical
      shifts not correlated between nuclei
    More information to identify signals
    Lower sensitivity to MW of protein
 Double-Resonance Experiments
Increases Resolution/Information Content
                          15N-1H   HSQC

      R             R
-15N - Ca- CO -15N - Ca
  H             H
Large Scalar Couplings  Less
Sensitive to MW of the Protein

                  Superior to 1H
                 homonuclear NMR:
                 all JH-H < 20 Hz
                  Mixing is faster so
                 less time for signal to
                 relax

								
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