NMR - University of Puget Sound

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NMR for structural biology
purification
mg protein
Protein structure possible since 1980s, due
to 2-dimensional (and 3D and 4D) NMR
Kurt Wuthrich (Nobel prize 2002)
JG/10-09
Protein domain
from a database
DNA
NMR structure determination steps
•
NMR experiment
•
Resonance assignment (connect the spin
systems with short-range NOEs)
•
Structural restraints
•
•
•
Structure calculations
•
•
JG/10-09
Distances (from NOEs)
torsion angles (from J coupling)
Conformation of polypeptide that satisfies all
distance restraints
Structure validation (cross-check your data)
Step 1: The resonance assignment puzzle
750 MHz 1H NMR spectrum of the SH3 domain
of the tyrosine kinase FYN
Hydrogen atoms
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Ribbon representation
Solutions to the Challenges
• Increase dimensionality of spectra to better
resolve signals: 1234
• Detect signals from heteronuclei (13C,15N)
•
•
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Better resolved signals, different overlaps
More information to identify signals
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 <5Å distances (H-H)
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Step 2: Structure calculation using distance
matrix
•
•
Nuclear Overhauser Effect (NOE)
crosspeaks for all protons <5Å apart
(through space!)
With assignments, use NOEs to
calculate structure of protein
(distance matrix)
Tertiary Structure
Sequential
Intraresidue
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A
B
C
D
Medium-range
(helices)
••••
Z
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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A folded protein is
easily recognized, and
H/D exchange tells
about hydrogen bond
stability
JG/10-09
Protein
domain
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)
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
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?
exterior face
interior face
Double-resonance experiments
increase 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
protein…aggregation!)
R
R
-15N-Ca-CO-15N-Ca H
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H
15N-1H
HSQC
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