NMR for structural biology

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NMR for structural biology
Protein domain from a database
purification
mg protein
Protein structure possible since 1980s, due to
2-dimensional (and 3D and 4D) NMR Kurt Wuthrich
(Nobel prize 2002)
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NMR structure determination steps

• NMR experiment
• Resonance assignment (connect the spin systems
  with short-range NOEs)
• Structural restraints
• Distances (from NOEs)
• torsion angles (from J coupling)
• Structure calculations
• Conformation of polypeptide that satisfies all
  distance restraints
• Structure validation (cross-check your data)

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Step 1 The resonance assignment puzzle
750 MHz 1H NMR spectrum of the SH3 domain of the
tyrosine kinase FYN
Ribbon representation
Hydrogen atoms
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Solutions to the Challenges

• Increase dimensionality of spectra to better
• resolve signals 1?2?3?4
• Detect signals from heteronuclei (13C,15N)
• Better resolved signals, different overlaps
• More information to identify signals

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HNCO
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Step 1 2D COSY spectrum. Protons transfer
magnetization to 3-bond coupled protons
(J-coupling)
Crosspeaks at chemical shift of correlated
protons.
Assign amino acid spin systems (3-bond coupled
protons)!
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2D NOESY spectrum through-space lt5Å distances
(H-H)
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Step 2 Structure calculation using distance
matrix

• Nuclear Overhauser Effect (NOE)
• crosspeaks for all protons lt5Å apart
• (through space!)
• With assignments, use NOEs to
• calculate structure of protein
• (distance matrix)

Tertiary Structure
Sequential
Intraresidue
Medium-range (helices)
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NMR Structure ensemble

• 20 structures all satisfy observed NOE (distance)
  data
• Some regions of protein are more dynamic, and the
  NMR structures show the range of conformations
  that the protein samples.

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Protein domain
A folded protein is easily recognized, and H/D
exchange tells about hydrogen bond stability
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Globular protein tertiary structure
Sidechain location vs. polarity -Nonpolar
residues in interior of protein (hydrophobic
effect promotes this, as well as efficient
packing of those sidechains) -Charged polar
residues on protein surface (immersing charge in
anhydrous interior is energetically
unfavorable) -Uncharged polar groups occur in
both places (hydrogen bonding and electrostatic
interactions inside the protein neutralize
their polarity)
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Amphipathic helices, sheets Protein interiors
compact (more efficient packing than organic
molecule crystals!) -however, they have
low-energy arrangements of sidechains (no
steric clashes) -exclude water (where present it
often makes specific H-bond bridge) -maximize
vdW surface complementarity These low-energy
characteristics have evolved(If a more stable
protein helps it function and the organism
survive, then the amino acids conferring the most
stability will be selected for.)
a-helix
b-sheet
purple nonpolar
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Globular protein structure4
Sample problems 1) Estimate the number of amino
acids in this protein. 2) What are reasonable
amino acid sidechains for the inner and outer
faces of these helices? (Just do the first three
turns of the red one) Draw or name 3 interior
and 3 exterior a.a. for each helix. 3) What a.a.
sidechain(s) can coordinate the heme iron atom?
interior face
exterior face
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Double-resonance experimentsincrease
resolution/information content
Correlates proton chemical shifts with chemical
shifts of other NMR nuclei such as 15N (needs
labeling!) or 13C (1.1 natural
abundance, possible with 100mM fumarate, but not
proteinaggregation!)