Protein NMR
Nuclear Magnetic Resonance
• NMR is an analytical technique in which
magnetic nuclei absorb energy from an
applied electromagnetic pulse and radiate the
energy back
• NMR is used for identifying functional groups
and imaging
Quantum theory
• 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 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 can 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
Chemical Shift
• When the magnetic field at the nucleis is not equal to
the applied magnetic field, the nucleus is sheilded by
surrounding electrons.
• Sheilding: If electrons produce a field opposing the
applied magnetic field, the applied field strength must
increase in order to reach the transition frueqncy
– This results in upfield shift
• Desheilding: If the electrons produce a field with the
applied magnetic field, the applied field strength must
decrease in order to reach the transition frequency.
– This results in downfield shift
Protein Structure
• 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
Protein 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
Myoglobin from E.Coli
(PDB ID: 1CQ2)
Protein NMR
• Series of 2D NMR
techniques
• First is usually 15N-HSQC
which results one signal
per amino acid residue
• Additional peaks from
amino acids containing
nitrogen in their side
chains
Diagram of Interactions study by other NMR
techniques including HNCO (b), HNCA (c),
HBHA(CBCACO)NH (d) and CBCANH (e)
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.
• Mainly show H-N correlations
– Amino acids with NH bonds in side
chains also have additional peaks
Transfer of Energy between
an excited hydrogen nuclei
to a neighboring 15N.
Advantages & Disadvantages
• Advantages
– “Fingerprint”: each protein has a unique pattern
– Identification of possible problems due to multiple
conformation of sampel heterogenity
• Disadvantages
– Can not assign specific peaks to specific atoms
– Need further more expensive analysis
Coupling Enhanced 750 Hz HSQC spectrum. Note that there are two peaks on a column
for each amide site. Variations can be seen in assigned doublets (veritcal lines). Most
variations are small compared to typical 15N line width.