JG/10-09
NMR for structural biologyNMR for structural biology
DNADNApurificationpurification
Protein Protein domain from a domain from a
databasedatabase
Protein structure possible since 1980s, due to 2-dimensional (and 3D and 4D) NMRKurt Wuthrich (Nobel prize 2002)
mg proteinmg protein
JG/10-09
NMR structure determination stepsNMR 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)
JG/10-09
Step 1: The resonance assignment puzzleStep 1: The resonance assignment puzzle
Ribbon representationHydrogen atoms
750 MHz 1H NMR spectrum of the SH3 domain of the tyrosine kinase FYN
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Solutions to the ChallengesSolutions to the Challenges
• Increase dimensionality of spectra to better
resolve signals: 1234
• Detect signals from heteronuclei (13C,15N)
• Better resolved signals, different overlaps
• More information to identify signals
JG/10-09
HNCOHNCO
JG/10-09
Crosspeaks at chemical shift of correlated protons.
Assign amino acid spin systems (3-bond coupled protons)!
Step 1: 2D COSY spectrum. Protons transfer Step 1: 2D COSY spectrum. Protons transfer magnetization to 3-bond coupled protons magnetization to 3-bond coupled protons (J-coupling)(J-coupling)
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2D NOESY spectrum: through-space <52D NOESY spectrum: through-space <5Å distances (H-H)Å distances (H-H)
JG/10-09
• Nuclear Overhauser Effect (NOE)crosspeaks for all protons <5Å apart(through space!)
• With assignments, use NOEs to calculate structure of protein (distance matrix)
Intraresidue
Sequential
Medium-range(helices)
A B C D Z• • • •
Tertiary Structure
Step 2: Structure calculation using Step 2: Structure calculation using distance matrixdistance matrix
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NMR Structure ensembleNMR 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.
JG/10-09
A folded protein is easily recognized, and H/D exchange tells about hydrogen bond stability
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 anhydrousinterior 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 stericclashes)
-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.)
-helix-sheetpurple nonpolar
Globular protein structure4
interior faceexterior face
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?
JG/10-09
15N-1H HSQC HSQC
-15N-C-CO-15N-C -
H
R
H
R
Double-resonance experimentsDouble-resonance experimentsincrease resolution/information contentincrease resolution/information content
Correlates proton chemicalshifts with chemical shiftsof other NMR nuclei suchas 15N (needs labeling!) or 13C (1.1% natural abundance,possible with 100mM fumarate, but not protein…aggregation!)
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