NMR Spectroscopy

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NMR Spectroscopy Part 2

• Judith Klein-SeetharamanDepartment of Structural
  Biology
• jks33_at_pitt.edu

2
NMR parameters
Chemical Shift
H2O
methyl
aromatic
Trp-side-chain NH
OH
aliphatic
Backbone NH
Side-chain HN
Ha
Spectrum see handout
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1d 1H NMR spectra
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NMR of membrane proteins
In detergent micelle
In lipid bilayer
http//www.elmhurst.edu/chm/vchembook/558micelle.
html
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Problems!
Detergent peaks
Detergent signals cause dynamic range
problems (Detergent signals cause spectral
overlap) Detergent deuteration is often not
feasible
Problem 1H,1H NOESY spectra do not show protein
signals
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Selective excitation
B. Selective excitation of the same region as in
A. Using excitation sculpting.
A. Selective excitation of the NH region using 90
degree pulse followed by direct observation.
Backbone NH
Tryptophan side chain NH
20
15
10
5
10
5
0
-5
1H Chemical Shift ppm
1H Chemical Shift ppm
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2d HSQC
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1d projection of HSQC
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HSQC spectra
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Chemical shift perturbation
Figure 2 in Cap-free structure of eIF4E suggests
a basis for conformational regulation by its
ligands Laurent Volpon, Michael J Osborne, Ivan
Topisirovic, Nadeem Siddiqui and Katherine LB
Borden The EMBO Journal (2006) 25, 51385149
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Assignment is needed!
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Several different assignment strategies exist
Most easily automated

• HNCO
• HNCOCACB
• HNCOCA
• HNCACB
• HNCA
• HNCACO

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Experiments
Figure 9. HNCO experiment. The magnetization is
transferred (blue arrows) from the HN(i) proton
via the N(i) atom to the directly attached
CO(i-1) carbon atom and returns the same way to
the HN(i) nucleus which is directly detected. The
frequencies of all three nuclei (red) are
detected. Image and description downloaded from
http//www.cryst.bbk.ac.uk/PPS2/projects/schirra/h
tml/3dnmr.htm
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Experiments
Figure 14. HNCA experiment. The HNCA experiment
is the prototype of all triple resonance
experiments. Starting at an amide proton (H) the
magnetization is transferred to the directly
attached nitrogen atom (N) which is measured as
the first spectral dimension. Then the
magnetization is transferred to the Calpha
nucleus (CA) which is measured as second
dimension. Afterwards, the magnetization is
transferred back the same way to the amide proton
which is measured as the third (direct)
dimension. Image and description downloaded from
http//www.cryst.bbk.ac.uk/PPS2/projects/schirra/h
tml/3dnmr.htm
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Experiments
Figure 15. HNCACO experiment. In the HN(CA)CO
experiment the magnetization is transferred from
the HN(i) proton via the N(i) atom and the CA
nucleus (Calpha(i)) to the CO(i) carbon atom and
back the same way. The Calpha atom (yellow) acts
only as relay nucleus, its frequency is not
detected. It is only the frequencies of HN, N and
CO (red) which are detected. Image and
description downloaded from http//www.cryst.bbk.a
c.uk/PPS2/projects/schirra/html/3dnmr.htm
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Assignments
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Structure Prediction by NMR
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NMR parameters

• chemical shifts
• NOE
• Dipolar coupling
• Scalar coupling constants (gives dihedral angles)
• Solvent exchange
• HetNOE
• longitudinal relaxation rates (R1)
• transverse relaxation rates (R2)

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NMR parameters
The Nuclear Overhauser Effect
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
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HSQC TOCSY
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
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NMR Parameters
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Dipolar Couplings
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Scalar coupling constants
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Structure Calculations

• Distance geometry
• Determines ensembles of structures consistent
  with an incomplete set of distance restraints
• Metric matrix algorithm
• Variable target function approach
• Restrained molecular dynamics
• Cartesian or torsion-angle coordinate systems
• Molecular dynamics force fields are supplemented
  by pseudo energy terms based on the NMR-derived
  restraints

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Structure Prediction From NMR Parameters
Most widely used software suites

• CNS
• http//cns.csb.yale.edu/v1.1/
• XPLOR
• http//xplor.csb.yale.edu/xplor/

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NMR parameters

• chemical shifts
• NOE
• Dipolar coupling
• Scalar coupling constants (gives dihedral angles)
• Solvent exchange
• HetNOE
• longitudinal relaxation rates (R1)
• transverse relaxation rates (R2)

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Comparison of T1 and T2 relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
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Dynamics in folded/unfolded lysozyme
Unfolded
Folded
Smaller rates more flexible