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

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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)

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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

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HNCOHNCO

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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)

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• 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.

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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?

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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!)