NMR Spectroscopy

Judith Klein-Seetharaman
Department of Structural Biology
jks33@pitt.edu
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)
 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/laureates/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/webcourse/
• http://spincore.com/nmrinfo/
• http://www.chembio.uoguelph.ca/driguana/NMR/TOC.HTM
• http://www.embl-heidelberg.de/nmr/sattler/embo/handouts/griesinger_lecture_pof.pdf
• http://dupont.molbio.ku.dk/teach/course/introNMR.pdf
• http://www.infochembio.ethz.ch/links/en/spectrosc_nmr_lehr.html
 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
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)
Nuclei in a magnetic field
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Energy Difference
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Macroscopic View
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Experiment: Recycle delay dependent on T1
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

relaxation
 The NMR signal
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

• Analogy: conducting loop rotating in a magnetic

field:
Fourier Transform
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Soft pulses vs. hard pulses
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Obtaining a spectrum
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Product Operator Formalism
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
HSQC Experiment
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
HSQC TOCXY
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
 Signal Intensity
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
• Boltzmann distribution
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)
 Sample requirements: Sources
Think of the requirements that we may need to fulfil!
C-tail

C-II
C-I
C-III VIII
105
VI
VII

IV 110
V

II I 115
III
120

4 125
3
E-II
E-I 130
2
1 5

E-III 1

N-tail 12 11 10 9 8 7 6
1
H C h e m i c a l S h i ft ( p p m )
 Example
Comparison of expression systems for rhodopsin

spacebio.net/modules/ mb_teare.html genetics.med.harvard.edu/ ~winston/ http://www.icr.ac.uk/structbi/baculovirus/i http://www.gla.ac.uk/ibls/BMB/mdh/images/conrd1 http://www.wjgnet.com/images/english/V11/257

mg/infectedsf9.jpg -cos-golgi.gif 6-2a.jpg

___________ ___________ ___________ ___________ ___________

___________

What are the advantages and disadvantages of each expression system?

 Where can we get these molecules from?
C-tail

C-II
C-I
C-III VIII

VI
VII

IV
V

II I

III

4
3
E-II
E-I
2
E-III 1

N-tail
 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
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)
NMR parameters
Chemical Shift http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

See handout
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, 5138–5149
 NMR parameters
The Nuclear Overhauser Effect
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Measuring NOE’s
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
NMR Parameters
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Dipolar Couplings
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 ID:1G5J

Drug design approach: SAR by NMR

Identify a drug that binds in the BH3 pocket of Bcl-XL, inhibit binding to BID ->
normal apoptosis.
 NMR parameters
• chemical shifts
• NOE
• Dipolar coupling 105

N C h e m ic a l S h ift ( p p m )
• coupling 110

constants 115

• HetNOE 120

• longitudinal 125

relaxation rates 130

15

(R1) 12 11 10 9 8 7 6
• transverse 1
H C h e m ic a l S h ift ( p p m )
relaxation rates
(R2)
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)
 Theory
-NMR Relaxation mechanism

NMR Dynamics on Different Time

Scales
time scale type
ns-ps fast internal motions
us-ms slow internal motions
Protein are dynamic molecules ms-days proton exchange

http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/relaxdyn

NMR dynamics can be used on a broad range of timescales.

 Relaxation

• Longitudinal relaxation (T1): return of longitudinal

(z-component) to its equilibrium value
• Transverse relaxation (T2): decay of transverse
(x,y-component)
T1 Relaxation
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Experiment: Recycle delay dependent on T1
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

relaxation
T2 Relaxation
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
 Mechanisms of Relaxation
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

• Dipolar interaction
 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
 Quantification of motion
- strategies to obtain dynamic information from NMR relaxation experiment

Measure R1, R2, heteronuclear NOE

“model free” approach

Get order parameter S2 ,τe, τm

Lipari-Szabo Model Free Approach
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Lipari Szabo
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

• Order parameters S2
• τe, effective correlation function
time for internal motions
• τm, overall tumbling correlation
time for global motions
 Lipari–Szabo “model-free” approach
- don’t depend on a specific physical model

• Estimate (τm) 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, 408–421 (1999)
“Model-free” is the most popular method to calculate dynamic parameters
Inversion Recovery: Measure T1
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Carr-Purcell spin echo: Measure T2
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Comparison of T1 and T2 relaxation
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Dependence of T1, T2 on Tumbling Time
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Chemical Exchange
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Chemical Exchange
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
 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
Dynamics in folded/unfolded lysozyme
Unfolded: Arrows indicate oxidized (all disulfide bonds present) lysozyme

Folded:
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)
Chemical shift
differences
between
unfolded
lysozyme and
random coil
 Relaxation Rates in Unfolded Lysozyme

Urea Water
12
10
8
R2 [s-1]

6
4
2
0
0 25 50 75 100 125 0 25 50 75 100 125
Residue Residue

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.
Relaxation Rates in Unfolded Lysozyme

Urea Water
12
10
8
R2 [s-1]

6
4
2
0
0 25 50 75 100 125 0 25 50 75 100 125
Residue Residue

What do you observe?

 Relaxation Rates in Unfolded Lysozyme

Urea Water
12
10
8
R2 [s-1]

6
4
2
0
0 25 50 75 100 125 0 25 50 75 100 125
Residue Residue

Regions with higher relaxation rates are localized as clusters.

 Presence of clusters of residual structure that are restricted in

conformational space, thus relax faster.
 Analysis of the relaxation data

Three means of analysis have been proposed:

a. Model-free approach
b. Cole-Cole distributions
c. Gaussian clusters

However: What gives rise to these clusters is not known.

 Relaxation Rates in Unfolded Lysozyme

Urea Water
12
10 3.
8 2. 5.
R2 [s-1]

6
4. 6.
1.
4
2
0
0 25 50 75 100 125 0 25 50 75 100 125
Residue Residue

Random Coil Model of Segmental Motion + Gaussian Distributions of Deviations

2
N |i  j | |i  x0 |
 
R (i )  Rint rinsic  e 
  Ae b

j 1 x0

There are six clusters of residual structure in HEWL-SME.

 Mapping of residual structure on the
native structure

How would you test what stabilizes these clusters?

Hydrophobic clusters of residual structure
How would you test for the presence of long-
range interactions?
Approach: Study effect of mutation
Effect of mutation on chemical shifts
 Effect of mutation on relaxation rates

A. Wild-Type B. W62G
12

10 2.
3.
8
5. 6.
R2 [s-1]

6 4.
1.
4

0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Residue Residue

A single point mutation, W62G in cluster 3, disrupts all

clusters in reduced and methylated lysozyme.
Effect of mutation on chemical shifts
 Effect of
mutation on
relaxation rates
Model for unfolded ensemble
Compactness
by NMR
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)
 NMR spectroscopy
General limitations

• Size
• Stability
• Sample homogeneity
• Need for labeling
• Quantities and source of biomolecules
 Resolution
Wide versus small chemical shift dispersion

folded unfolded

112
105

N C h e m ic a l S h ift (p p m )
N C h e m ic a l S h ift ( p p m )

114
110 116
115 118
120
120
122
125 124
126
130

15
128
15

12 11 10 9 8 7 6 1 0 .0 9 .0 8 .0 7 .0
1
1
H C h e m ic a l S h ift ( p p m ) H C h e m ic a l S h ift ( p p m )

Unfolded proteins have a small chemical shift dispersion.

1d 1H NMR spectra
HSQC spectra
 Example Solution NMR of DAGK
1
H,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: S24–S40
Example Solution NMR of Rhodopsin
It’s a headache.
 What is signal 1?
How can you test your hypothesis?
 Assignment of Signal 1
NMR Spectroscopy
Black: original spectrum,
An enzyme was
red: C-terminus, green: N-
used to cleave off
terminus (after AspN
the C-terminus at
cleavage)
the site indicated
below:
 Traditional Solution NMR Approaches
Problems with full-length membrane proteins in detergents

1. Size – is not the only problem

A.
(Trosy does not work for helical

Chemical Shift (ppm)

132 126 120 114 108
NH2

 membrane proteins)
2. Conformational exchange –
fluctuations in the detergent
 micelle environment lead to fast
relaxation thus signal decay
15N
12.0 11.0 10.0 9.0 8.0 7.0
3. Spin diffusion – cannot deuterate
B.
Chemical Shift (ppm)
samples from mammalian cells
132 126 120 114 108


15N

Problem: Traditional assignment

12.0 11.0 10.0 9.0 8.0 7.0
1H Chemical Shift (ppm)
strategies using triple resonance
Klein-Seetharaman et al. (2004) PNAS 101, 3409-13.
experiments (13C,15N,1H) don’t work
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

 Evidence I: Selective excitation

A. Selective excitation of the NH region using B. Selective excitation of the

90 degree pulse followed by direct same region as in A. Using
observation. excitation sculpting.

Backbone NH

Tryptophan side chain NH

20 15 10 5 10 5 0 -5
1
H Chemical Shift [ppm] 1
H Chemical Shift [ppm]
You might also want to develop your own
biophysical approaches…
 19
F NMR Spectroscopy
Approach

Advantages of 19F NMR • no natural background in proteins

• high sensitivity
• sensitive to differences in environment
General Method for Attachment of 19F Label
Rho SH
+Dithiodipyridine

I II III IV V VI VII
Rho S S N -
348
OOC - A PAV Q S T ET K S V T
T SA
248 ED
245 C 316
Cytoplasmic S N FR C Q E S
311 D
M C M V G
67
C P F
G Q TA N T C L
K 140 A Q K QT T
L C 150 E A R P
KK R A F N
Disulfide
65
V A E Q L
H Q T VV H
N KE E K 250 N K CCG
K
TV V IA M T V M R TV MM
CF3 CH2 SH
P
Y L ER Y VF L V I
TL N
I Y VL
AI VG A G Q I I I Y
L M PV
Exchange N F
I
LL N
G F P 50 A A V
L S LV
LW
W TF
AM L
V F F CY A I V
I VI I L
F
300 Y N
V
(TET) IM
L
L
L
LD
V M F
IA
G G E A CA
A I I P L P L WC K TS
Y F F A
A

MF GF TL PP L V H F GA A Membrane
FG FA GV AV TIP
AA
Y
T T G F W Y MFV YF M
L T E V I I
SM L 110 N L S F
STY GC R SF F I
W QF L Y E T P
T I G
EP 100 H P 200 N HQ
A G G
P
E 187 N TE GS D F
LYY P AEF YF V F G C E Intradiscal
Q
Rho S S CH2 CF3
PS M Q C S GI H
R V V GT K DY Y T P
1 G TE N
AcN- M N GP S
NF
YV PF

Large-scale expression system for rhodopsin: HEK293 cells

 19
F NMR Spectroscopy
Qualitative Changes
dark light
Dark
10.2 ppm Light (3')
10.6 ppm
I. Cys-140 23'
43'
10.1 ppm
10.0 ppm 63'
II. Cys-316

9.9 ppm 9.9 ppm

III. K311C

9.9 ppm 10.5 10.0 9.5 9.0 8.5

9.9 ppm
IV. K245C
Chemical shift (ppm relative to TFA)

9.8 ppm 9.6 ppm

V. K67C

9.5 ppm
9.6 ppm
VI. K248C

11.5 11.0 10.5 10.0 9.5 9.0 8.5

Chemical shift (ppm relative to TFA)
 Proximity
19
F-19F Nuclear Overhauser Effect

S S CH2 CF3

S S CH2 CF3
Rho
Magic angle spinning
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Enhancing resolution in solid state NMR
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

Aligned: Magic angle spinning:

Current Status of solid state NMR
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
REDOR
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
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)
 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
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
The Shim System
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Monitoring Shimming
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
Monitoring shimming
http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf
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!!
 Outlook for next week

• Lecture: The structure determination

pipeline
• Practical analysis of NMR data in
computer lab:
– Topspin
– NMRpipe
– NMRviewJ