NMR

NMR

1. Benefits

   • Probe molecules in their "natural" state
     • Structure
     • Function
     • Dynamics
     • Interactions with ligands, proteins, membranes etc
   • No need to crystallise
     • No crystal packing effects
   • Applications to "natively unstructured" proteins

2. History

   • Discovery 1946
     • Bloch, Stanford
     • Purcell, Harvard
     • Ribonuclease A first protein in 1953
     • Nobel Prize for physics 1953
     • Modern FT-NMR method developed 1992
       • Ernst, Zurich
         • Nobel Prize for chemistry
     • TROSY method for large proteins developed 1997
     • NMR in protein structure determination 2002
       • Wuthrich, Tanaka and Fenn
         • Nobel Prize for chemistry

3. ·

   • Peptide bonds
     • Carbonyl carbom = CO or C'
     • C-alpha bears the side chain
     • Side chains labelled with Greek letters
       • ' denotes equivalent atoms

4. Basic principles

   • Spin
     • Intrinsic property of nucleus
       • Originates from quantum mechanics
         • Defined by quantum number I
           • Z-component of I is labelled Iz and is quantised
       • 1H, 31P, naturally abundant
       • 13C, 15N, naturally less abundant
         • Can be incorporated through isotope labelling
     • Nuclear magnetic moment related to spin angular momentum
       • The link is the magnetogyric ratio (gamma)
     • Associated with magnetic moment
       • Required for determining NMR
       • Defines basic resonance frequency
     • Each spin in a molecule gives rise to an NMR line
       • Exact resonance frequency depends on chemical environment of each spin
       • Dependent on the atom type
         • Spin moments quantised
   • Allowed transitions obey the selection rule
     • Energetic
   • Chemical shift
     • The nucleus is "screened" by electrons
       • So it experiences a local field
         • This is quoted as a shift from the reference signal
           • Measured in ppm
     • Reference
       • 1-H
         • TMS at 0ppm
           • As not soluble in water use 4,4-dimethyl-4-silapentane-1-sulfonic acid
       • 13-C
         • TMS at 0ppm
           • Generally referenced indirectly from 1H
       • 15-N
         • Liquid ammonia 0ppm
           • Nitromethane used in organic chemistry
             • Difference ~380ppm downfield
       • 31-P
         • 85% H3PO4 at 0ppm
       • Amino acids
         • Based on the general locations of H chemical shifts
           • Take an average for the reference, based on values for 13 proteins (Kraulis)

5. NOE

   • Nuclear overhauser effect
     • The most important structural information derived from NMR
     • Result of cross-talk between different spins
       • Dependent on through-space distance between spins
         • Tend to be only observed when protons are separated by less than 5-6A
   • Other effects
     • J-coupling constants
       • Information about dihedral angles
         • Define peptide backbone and side chain conformations
     • New NMR parameters
       • Residual dipolar couplings
         • Measured in anisotropic solution
         • Extremely useful in defining relative orientation of two protein domains
           • NOEs often not observed, too far apart
       • Cross-correlated relaxation effects
       • Shown to provide distance-independent projection angles for bond vectors

6. Applications to structural biology

   • Membrane proteins
     • Solid state
     • Typically don't crystallise
   • MRI
     • In vivo tissue imaging
     • CT
       • Slice images of the body
         • Localised spectroscopy
           • Study metabolism in different tissues or organs
   • Liquid-state NMR
     • Uses
       • Constitution/conformation of organic molecules
       • 3D structures
         • Proteins
         • Nucleic acids
         • Sugars
       • Conformational dynamics/mobility of biomacromolecules in solution
         • Mobility of peptide backbone
         • Populations of amino acid side chain conformations
         • DNA/RNA base pair dynamics
       • Chemical and conformational exchange
         • Hydrogen bonding
         • Base pair opening dynamics
       • Enzyme mechanisms
       • Chemical reactions
       • Dynamics/lifetimes of bound water molecules
       • Folding studies
       • Rational drug design
   • Need 5-10mg for a 20kDa molecule currently
     • New cytoprobe technology needs ~2mg
   • 1D NMR takes 5 minutes
     • Determines whether protein has 3ry structure

7. Energy, populations and signal-to-noise

   • Spin states
     • Alpha
       • Spins precess around axis of static magnetic field with Z-axis projection parallel to it
     • Beta
       • Spins precess around axis of static magnetic field with Z-axis projection antiparallel to it
     • Population of these states almost equal
       • According to Boltzmann distribution
       • Energies involved are fairly small
     • No magnetisation in x,y plane
       • Individual precession frequencies uncorrelated
         • Out of plane
           • x or y components average zero
     • Measuring precession frequencies
       • System disturbed
         • Brought into non-equilibrium
           • Monitor transverse (x or y) magnetisation
   • Resonance frequencies
     • Correspond to radio frequency wavelengths
       • Low frequency
       • Separation of energy levels defines population difference
         • Provides magnetisation measured in NMR experiment
   • Relatively insensitive
     • Optimising signal-to-noise ratio is critical
       • Use more spins
         • Higher sample concentration
       • Higher strengths for static magnetic field
       • Use highest energy spins for excitation and detection

8. Inducing resonance

   • Oscillatory magnetic field applied
     • Frequency corresponding to separation of two spin energy levels
       • Varies due to chemical shifts
         • A number of frequencies are applied
           • Two methods
             • Vary within expected range like tuning in a radio
               • Continuous wave
                 • Takes time for step-wise tuning over a range
             • Very short frequency
               • Inherently encodes a range of frequencies
                 • Induce resonance for the whole frequency spectrum in one experiment
                   • Fourier transform
                     • Response is a superposition of frequencies of all spins as a function of time F(t)
                       • Fourier transformation creates corresponding spectrum: F(omega) intensity as a function of frequency
                         • Converts a function in the time domain into the frequency domain
                   • All at once
                     • More sensitive
     • Relaxation and NMR linewidths
       • After applying 90-degree pulse to equilibrium z-magnetisation, transverse magnetisation is created
         • Correlates phases of individual spin precession frequencies
         • Non-random superposition of states
           • Coherence
             • Population difference between alpha and beta states has disappeared
               • This corresponds to Z-magnetisation
                 • System in non-equilibrium
                   • Enter text here

9. NMR sample preparation

   • Overexpress POI in E. coli
     • Prepare isotopically labelled samples with 13C and 15N
       • Molecule >25kDa
         • Deuterium labelling
     • Obtain RNA molecules from in vitro transcription
       • Try constructs of varying size and domain boundaries
         • Creates samples which give good NMR spectra

10. Calculating structure

    • Record heteronuclear multidimensional NMR experiments
      • Assignment of all chemical shifts
    • Measure 13C and 15N 3D NOESY spectra
      • Obtain structural information based on interproton NOEs
      • Assignment of interatomic distances is time consuming
        • Can be semi-automated; need to know chemical shift data
    • Total: ~4 weeks
    • Spectra acquired using Fourier transform NMR
      • Need to use Fourier transformation in order to obtain frequency data
        • Can improve signal-to-noise during this processing
    • 3J coupling constants and residual dipolar couplings give additional structural information
    • Restrained molecular dynamics/simulated annealing structure calculation
    • Iterative
      • Combine a large number of inputs
        • Should converge to the same fold
    • Spectral assignment
      • Often the first piece of information
        • NMR difficult without
      • Strategy depends on size
        • Small <10 kDa
          • Homonuclear
            • Proton shifts only
              • No H-H coupling across peptide bond
            • COSY/TOCSY
              • Through bond correlations
            • NOESY
              • Through space correlations
                • Measures connections between amino acids
                  • "walk" along protein
                  • Detect distances
        • Medium <25 kDa
          • Heteronuclear
            • Isotopic labelling
            • Mostly 3D experiments
              • A cube of data viewed as slices
            • HSQC
              • 2D
                • Filtered NOESY and TOCSY
        • Large >25 kDa
          • TROSY
          • Deuteration
    • Dimensions
      • Increase complexity
        • Experiment takes longer

11. General protocol

    • 1. Identify amino acid spin systems
      • COSY
      • TOCSY
    • 2. Obtain sequential connectivities
      • Backbone NOE analysis
    • 3. Extend assignments to complete spin systems
      • Side chains
        • TOCSY
        • NOESY