Protein Nuclear Magnetic Resonance (NMR)

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Quantum Theory
Spin
  • Subatomic particles “spin” along their axis.
  • Atoms with paired spins have no net overall spin.
  • Atoms can have a net overall spin if the number of protons or the number of protons and neutrons is odd.
    • These atoms have multiple orientations
    • Examples: 13C, 15N
  • In the absence of an external magnetic field, these orientations have the same energy.
  • In the presence of an external magnetic field, the energy level splits
  • The lower energy level contains more nuclei that the higher energy level. 
  • Lower energy nuclei be excited into the higher energy level by electromagnetic radition.
  • The frequency of the radition corresponds to the energy difference between the two nuclei states (transition frequency). 
  • After absorption, nuclei relax to lower energy state

Energy Level Diagram.  Without an external magnetic
field, orientations have the same energy.  In the presence
of an applied magnetic field, the energy level splits into
two levels, high energy and low energy. 
source: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm

Chemical Shift
Protein Structure
 
Primary Structure.  Peptide
bonds are highlighted in red. 

  • Primary structure is a sequence of amino acids linked by peptide bonds
    • Each amino acid has a unique side chain (R); there are 20 total
    • Peptide bonds formed by dehydration synthesis (highlighted in red)
    • Chemical interactions of R groups dictate higher structure
  • Secondary structure is the formation of alpha helicies and beta sheets
  • Tertiary structure is the 3D folding of the protein
  • Quaternary structure is the assembly of multiple protein subunits

Protein NMR

  • Protein NMR is a series of 2D NMR techniques
  • First is usually 15N-HSQC which results one signal per amino acid residue

Heteronuclear Single Quantum Coherence  (HSQC)
  • Hydrogen nuclei are excited and the energy is transferred to a neighboring 15N
  • The chemical shift is evolved on the nitrogen
  • Energy is transferred back to the hydrogen for detection.
  • This method mainly shows H-N correlations.  Additional peaks are seen from
  • amino acids containing nitrogen in their side chains

Transfer of Energy between
an excited hydrogen nuclei
to a neighboring 15N. 


Advantages
“Fingerprint”: each protein has a unique pattern
Identification of possible problems due to multiple
conformation of sample heterogenity
Disadvantages
Can not assign specific peaks to specific atoms
Need further more expensive analysis

Results
 
HSQC spectrum (left) for myglobin from E.coli (Toiman).  Ribbon structure (right) for the same protein (PDB: 1CQ2). 

Resources
Bax, A.  And Grzesiek, S.  "Methodological Advances in Protein NMR."  Accounts of Chemical Research. 36 (1993): 131-138

Tolman, J.R. et al.  “Nuclear magnetic dipole interactions in field-oriented protein: Information for structure determination in solution.”  Proc. Natl. Acad. Sci.  92 (1995): 9279-9283. 

http://www.protein-nmr.org.uk/spectra.html

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm