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NMR Spectroscopy

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Title: NMR Spectroscopy


1
NMR Spectroscopy
  • Judith Klein-SeetharamanDepartment of Structural
    Biology
  • jks33_at_pitt.edu

2
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

3
Resources
Websites
  • http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
  • http//www.bmrb.wisc.edu/
  • http//www.biochem.ucl.ac.uk/bsm/nmr/ubq/
  • http//nobelprize.org/nobel_prizes/chemistry/laure
    ates/2002/wutrich-lecture.pdf
  • http//www.cis.rit.edu/htbooks/nmr/
  • http//www.ch.ic.ac.uk/local/organic/nmr.html
  • http//www.spectroscopynow.com/
  • http//www.chem.queensu.ca/FACILITIES/NMR/nmr/webc
    ourse/
  • http//spincore.com/nmrinfo/
  • http//www.chembio.uoguelph.ca/driguana/NMR/TOC.HT
    M
  • http//www.embl-heidelberg.de/nmr/sattler/embo/han
    douts/griesinger_lecture_pof.pdf
  • http//dupont.molbio.ku.dk/teach/course/introNMR.p
    df
  • http//www.infochembio.ethz.ch/links/en/spectrosc_
    nmr_lehr.html

4
Resources
Books
  • NMR Books
  • Protein NMR Techniques (Methods in Molecular
    Biology) by A. Kristina Downing (Editor)
  • Protein NMR Spectroscopy Principles and Practice
    by John Cavanagh, Wayne J. Fairbrother, III,
    Arthur G. Palmer, Nicholas J. Skelton, Mark Rance
  • Spin Dynamics Basics of Nuclear Magnetic
    Resonance by Malcolm H. Levitt
  • Principles of Nuclear Magnetic Resonance in One
    and Two Dimensions by Richard R. Ernst, Geoffrey
    Bodenhausen, Alexander Wokaun
  • 200 and More NMR Experiments A Practical Course
    by Stefan Berger, Siegmar Braun
  • Basic One- and Two-Dimensional NMR Spectroscopy
    by Horst Friebolin
  • NMR Spectroscopy Basic Principles, Concepts, and
    Applications in Chemistry by Harald Günther
  • NMR Data Processing by Hoch
  • NMR The Toolkit by P. J. Hore, J. A. Jones, S.
    Wimperis
  • Nuclear Magnetic Resonance by P. J. Hore
  • NMR for Physical and Biological Scientists by
    Thoma Pochapsky
  • Understanding NMR Spectroscopy by James Keeler
  • NMR of Proteins (Topics on Molecular and
    Structural Biology) by G. M. Clore, A. M.
    Gronenborn
  • The Nuclear Overhauser Effect in Structural and
    Conformational Analysis
  • by David Neuhaus, Michael P. Williamson
  • Biophysics Books with chapters on NMR
  • Biophysical Chemistry Part II Techniques for
    the Study of Biological Structure and Function by
    Charles R Cantor, Paul R Schimmel
  • Principles of Physical Biochemistry by Kensal E
    van Holde, Curtis Johnson, Pui Shing Ho

5
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

6
Nuclei in a magnetic field
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
7
Energy Difference
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
8
Macroscopic View
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
9
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
10
Experiment Recycle delay dependent on T1
relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
11
The NMR signal
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
  • Analogy conducting loop rotating in a magnetic
    field

12
Fourier Transform
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
13
Soft pulses vs. hard pulses
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
14
Obtaining a spectrum
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
15
Product Operator Formalism
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
16
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
17
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
18
HSQC Experiment
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
19
HSQC TOCXY
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
20
Signal Intensity
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
  • Boltzmann distribution

21
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

22
Sample requirements Sources
Think of the requirements that we may need to
fulfil!
23
Example
Comparison of expression systems for rhodopsin
http//www.gla.ac.uk/ibls/BMB/mdh/images/conrd1-co
s-golgi.gif
http//www.wjgnet.com/images/english/V11/2576-2a.j
pg
http//www.icr.ac.uk/structbi/baculovirus/img/infe
ctedsf9.jpg
spacebio.net/modules/ mb_teare.html
genetics.med.harvard.edu/ winston/
___________
___________
___________
___________
___________
___________
What are the advantages and disadvantages of each
expression system?
24
Where can we get these molecules from?
25
Sources of biomolecules
Summary
  • Native sources
  • Best quality (correct fold, posttranslational
    modifications etc.)
  • Not always best quantity
  • Limitations in labeling
  • No mutants
  • Chemical synthesis
  • Good for small molecules
  • Not good for large proteins
  • Biosynthesis
  • A variety of expression systems exist, all with
    their advantages and disadvantages.
  • Required for isotope labeling for NMR

26
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

27
NMR parameters
Chemical Shift
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
See handout
28
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
29
NMR parameters
The Nuclear Overhauser Effect
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
30
Measuring NOEs
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
31
NMR Parameters
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Dipolar Couplings
32
NMR Structure of Bcl-XL Bound to BH3 Peptide
  • Structure was solved with a homolog of BH3 helix.
  • Protein-protein interaction groove was identified
    on anti-apoptotic Bcl-XL.

PDB ID1G5J
Drug design approach SAR by NMR
  • Identify a drug that binds in the BH3 pocket of
    Bcl-XL, inhibit binding to BID -gt normal
    apoptosis.

33
NMR parameters
  • chemical shifts
  • NOE
  • Dipolar coupling
  • coupling constants
  • HetNOE
  • longitudinal relaxation rates (R1)
  • transverse relaxation rates (R2)

34
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

35
Theory-NMR Relaxation mechanism
  • NMR Dynamics on Different Time Scales
  • time scale type
  • ns-ps fast internal motions
  • us-ms slow internal motions
  • ms-days proton exchange

Protein are dynamic molecules
http//www.bioc.aecom.yu.edu/labs/girvlab/nmr/cour
se/relaxdyn
NMR dynamics can be used on a broad range of
timescales.
36
Relaxation
  • Longitudinal relaxation (T1) return of
    longitudinal (z-component) to its equilibrium
    value
  • Transverse relaxation (T2) decay of transverse
    (x,y-component)

37
T1 Relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
38
Experiment Recycle delay dependent on T1
relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
39
T2 Relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
40
Mechanisms of Relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
  • Dipolar interaction

41
Mechanisms of Relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
  • Chemical shift anisotropy
  • Scalar relaxation (chemical exchange, rapid T1
    relaxation)
  • Quadrupolar relaxation
  • Spin rotation relaxation
  • Interaction with unpaired electrons

42
Quantification of motion- strategies to obtain
dynamic information from NMR relaxation experiment
  • Measure R1, R2, heteronuclear NOE
  • model free approach
  • Get order parameter S2 ,te, tm

43
Lipari-Szabo Model Free Approach
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
44
Lipari Szabo
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
  • Order parameters S2
  • te, effective correlation function time for
    internal motions
  • tm, overall tumbling correlation time for global
    motions

45
LipariSzabo model-free approach - dont
depend on a specific physical model
  • Estimate (tm) from R2/R1 for a selected subset of
    the residues
  • fits to the observed relaxation data using
    various regression variables
  • model-selection criteria are used to decide which
    choice is appropriate for each residue
  • Reoptimize using the selected models.
  • Uncertainties in the optimized parameters were
    obtained by Monte Carlo simulation.

Michael Andrec, Gaetano T. Montelione, Ronald M.
Levy Journal of Magnetic Resonance 139, 408421
(1999)
Model-free is the most popular method to
calculate dynamic parameters
46
Inversion Recovery Measure T1
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
47
Carr-Purcell spin echo Measure T2
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
48
Comparison of T1 and T2 relaxation
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
49
Dependence of T1, T2 on Tumbling Time
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
50
Chemical Exchange
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
51
Chemical Exchange
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
52
Example GCN4-58 complexed with DNA-dynamics of
complex formation
dynamics of the basic leucine zipper domain of
the dimeric yeast transcription factor GCN4
(GCN4-58) as it relates to DNA binding
  • low S2 indicate high flexibility. S2 can be used
    to estimate energetics.

John Cavanagh and Mikael Akke nature
structural biology volume 7 number 1 january
2000
53
Dynamics in folded/unfolded lysozyme
Unfolded
Arrows indicate oxidized (all disulfide bonds
present) lysozyme
Folded
54
Using NMR to identify residual structure
  • Can in principle use all parameters
  • chemical shifts
  • coupling constants
  • HetNOE
  • longitudinal relaxation rates (R1)
  • transverse relaxation rates (R2)

55
Chemical shift differences between unfolded
lysozyme and random coil
56
Relaxation Rates in Unfolded Lysozyme
Unfolded lysozyme can be studied in 8 M
urea. Unfolded lysozyme can also be studied
without urea, if the disulfide bonds are reduced
and the cysteines are derivatized to prevent them
from forming disulfide bonds.
57
Relaxation Rates in Unfolded Lysozyme
What do you observe?
58
Relaxation Rates in Unfolded Lysozyme
Regions with higher relaxation rates are
localized as clusters. ? Presence of clusters of
residual structure that are restricted in
conformational space, thus relax faster.
59
Analysis of the relaxation data
  • Three means of analysis have been proposed
  • Model-free approach
  • Cole-Cole distributions
  • Gaussian clusters

However What gives rise to these clusters is not
known.
60
Relaxation Rates in Unfolded Lysozyme
There are six clusters of residual structure in
HEWL-SME.
61
Mapping of residual structure on the native
structure
How would you test what stabilizes these clusters?
62
Hydrophobic clusters of residual structure
63
How would you test for the presence of long-range
interactions?Approach Study effect of mutation
64
Effect of mutation on chemical shifts
65
Effect of mutation on relaxation rates
A single point mutation, W62G in cluster 3,
disrupts all clusters in reduced and methylated
lysozyme.
66
Effect of mutation on chemical shifts
67
Effect of mutation on relaxation rates
68
Model for unfolded ensemble
69
Compactness by NMR
70
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

71
NMR spectroscopy
General limitations
  • Size
  • Stability
  • Sample homogeneity
  • Need for labeling
  • Quantities and source of biomolecules

72
Resolution
Wide versus small chemical shift dispersion
folded
unfolded
Unfolded proteins have a small chemical shift
dispersion.
73
1d 1H NMR spectra
74
HSQC spectra
75
Example Solution NMR of DAGK
1H,15N-HSQC spectrum of a 120 aa long membrane
protein in DPC micelles
Diacylglycerol kinase
Charles R. Sanders, Frank Sonnichsen (2006)
Solution NMR of membrane proteins practice and
challenges. Magn. Reson. Chem. 2006 44 S24S40
76
Example Solution NMR of Rhodopsin
Its a headache.
77
What is signal 1?
How can you test your hypothesis?
78
Assignment of Signal 1
NMR Spectroscopy
Black original spectrum, red C-terminus, green
N-terminus (after AspN cleavage)
An enzyme was used to cleave off the C-terminus
at the site indicated below
79
Traditional Solution NMR Approaches
Problems with full-length membrane proteins in
detergents
  1. Size is not the only problem (Trosy does not
    work for helical membrane proteins)
  2. Conformational exchange fluctuations in the
    detergent micelle environment lead to fast
    relaxation thus signal decay
  3. Spin diffusion cannot deuterate samples from
    mammalian cells

Problem Traditional assignment strategies using
triple resonance experiments (13C,15N,1H) dont
work
Klein-Seetharaman et al. (2004) PNAS 101,
3409-13.
80
Traditional Solution NMR Approaches
Problems with full-length membrane proteins in
detergents
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
81
Evidence I 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
82
You might also want to develop your own
biophysical approaches
83
19F NMR Spectroscopy
Approach
  • no natural background in proteins
  • high sensitivity
  • sensitive to differences in environment

Advantages of 19F NMR
General Method for Attachment of 19F Label
Rho
SH
Dithiodipyridine
N
Rho
S
S
Disulfide
CH
CF
SH
2
3
Exchange
(TET)
Rho
CH
CF
S
S
2
3
Large-scale expression system for rhodopsin
HEK293 cells
84
19F NMR Spectroscopy
Qualitative Changes
dark
light
Dark
Light (3')
23'
43'
63'
8.5
9.0
9.5
10.0
10.5
Chemical shift (ppm relative to TFA)
85
Proximity
19F-19F Nuclear Overhauser Effect
CH
CF
S
S
2
3
CH
CF
S
S
2
3
Rho
86
Magic angle spinning
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
87
Enhancing resolution in solid state NMR
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Aligned
Magic angle spinning
88
Current Status of solid state NMR
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
89
REDOR
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
90
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)

91
The components of an NMR spectrometer
  • A magnet
  • Probehead(s)
  • Radiofrequency sources
  • Amplifiers
  • Analog to digital converters
  • The lock system
  • The shim system
  • A computer

92
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
93
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
94
The Shim System
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
95
Monitoring Shimming
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
96
Monitoring shimming
http//www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
97
Objectives of this Lecture and Practicum
  • Resources
  • Physical principle
  • Sample requirements
  • Parameters that are measured by NMR
  • Dynamics by NMR
  • Limitations
  • Practical aspects
  • Setup of NMR experiments (downstairs)
  • Bring your coats!!

98
Outlook for next week
  • Lecture The structure determination pipeline
  • Practical analysis of NMR data in computer lab
  • Topspin
  • NMRpipe
  • NMRviewJ
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