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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

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nmr spectroscopy

NMR Spectroscopy

Judith Klein-SeetharamanDepartment of Structural Biology

jks33@pitt.edu

objectives of this lecture and practicum
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

resources
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

resources4
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum5
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nuclei in a magnetic field
Nuclei in a magnetic field

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

energy difference
Energy Difference

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

macroscopic view
Macroscopic View

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide9

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

experiment recycle delay dependent on t1 relaxation
Experiment: Recycle delay dependent on T1 relaxation

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

the nmr signal
The NMR signal

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

  • Analogy: conducting loop rotating in a magnetic field:

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

fourier transform
Fourier Transform

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

soft pulses vs hard pulses
Soft pulses vs. hard pulses

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

obtaining a spectrum
Obtaining a spectrum

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

product operator formalism
Product Operator Formalism

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide16

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide17

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

hsqc experiment
HSQC Experiment

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

hsqc tocxy
HSQC TOCXY

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

signal intensity
Signal Intensity

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

  • Boltzmann distribution

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum21
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

sample requirements sources
Sample requirements: Sources

Think of the requirements that we may need to fulfil!

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

example
Example

Comparison of expression systems for rhodopsin

http://www.gla.ac.uk/ibls/BMB/mdh/images/conrd1-cos-golgi.gif

http://www.wjgnet.com/images/english/V11/2576-2a.jpg

http://www.icr.ac.uk/structbi/baculovirus/img/infectedsf9.jpg

spacebio.net/modules/ mb_teare.html

genetics.med.harvard.edu/ ~winston/

___________

___________

___________

___________

___________

___________

What are the advantages and disadvantages of each expression system?

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

where can we get these molecules from
Where can we get these molecules from?

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

sources of biomolecules
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum26
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nmr parameters
NMR parameters

Chemical Shift

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

See handout

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

chemical shift perturbation
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nmr parameters29
NMR parameters

The Nuclear Overhauser Effect

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

measuring noe s
Measuring NOE’s

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nmr parameters31
NMR Parameters

Dipolar Couplings

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nmr structure of bcl xl bound to bh3 peptide
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.

Identify a drug that binds in the BH3 pocket of Bcl-XL, inhibit binding to BID -> normal apoptosis.

PDB ID:1G5J

Drug design approach: SAR by NMR

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nmr parameters33
NMR parameters
  • chemical shifts
  • NOE
  • Dipolar coupling
  • coupling constants
  • HetNOE
  • longitudinal relaxation rates (R1)
  • transverse relaxation rates (R2)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum34
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

theory nmr relaxation mechanism
Theory-NMR Relaxation mechanism

NMR Dynamics on Different Time Scales

time scaletype

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/course/relaxdyn

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

relaxation
Relaxation
  • Longitudinal relaxation (T1): return of longitudinal (z-component) to its equilibrium value
  • Transverse relaxation (T2): decay of transverse (x,y-component)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

t1 relaxation
T1 Relaxation

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

experiment recycle delay dependent on t1 relaxation38
Experiment: Recycle delay dependent on T1 relaxation

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

t2 relaxation
T2 Relaxation

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

mechanisms of relaxation
Mechanisms of Relaxation

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

  • Dipolar interaction

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

mechanisms of relaxation41
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

quantification of motion strategies to obtain dynamic information from nmr relaxation experiment
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

lipari szabo model free approach
Lipari-Szabo Model Free Approach

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

lipari szabo
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

lipari szabo model free approach don t depend on a specific physical model
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, RonaldM. Levy Journal of Magnetic Resonance 139, 408–421 (1999)

“Model-free” is the most popular method to calculate dynamic parameters

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

inversion recovery measure t1
Inversion Recovery: Measure T1

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

carr purcell spin echo measure t2
Carr-Purcell spin echo: Measure T2

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

comparison of t1 and t2 relaxation
Comparison of T1 and T2 relaxation

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

dependence of t1 t2 on tumbling time
Dependence of T1, T2 on Tumbling Time

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

chemical exchange
Chemical Exchange

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

chemical exchange51
Chemical Exchange

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

example gcn4 58 complexed with dna dynamics of complex formation
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

dynamics in folded unfolded lysozyme
Dynamics in folded/unfolded lysozyme

Unfolded:

Arrows indicate oxidized (all disulfide bonds present) lysozyme

Folded:

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

using nmr to identify residual structure
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

chemical shift differences between unfolded lysozyme and random coil
Chemical shift differences between unfolded lysozyme and random coil

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

relaxation rates in unfolded lysozyme
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.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

relaxation rates in unfolded lysozyme57
Relaxation Rates in Unfolded Lysozyme

What do you observe?

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

relaxation rates in unfolded lysozyme58
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.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

analysis of the relaxation data
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.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

relaxation rates in unfolded lysozyme60

3.

2.

5.

4.

6.

1.

Random Coil Model of Segmental Motion

+ Gaussian Distributions of Deviations

2

-

-

|

i

x

|

|

i

j

|

N

0

-

-

å

å

=

+

l

R

(

i

)

R

e

Ae

b

int

rinsic

=

j

1

x

0

Relaxation Rates in Unfolded Lysozyme

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

mapping of residual structure on the native structure
Mapping of residual structure on the native structure

How would you test what stabilizes these clusters?

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

hydrophobic clusters of residual structure
Hydrophobic clusters of residual structure

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

how would you test for the presence of long range interactions approach study effect of mutation
How would you test for the presence of long-range interactions?Approach: Study effect of mutation

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

effect of mutation on chemical shifts
Effect of mutation on chemical shifts

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

effect of mutation on relaxation rates
Effect of mutation on relaxation rates

A single point mutation, W62G in cluster 3, disrupts all clusters in reduced and methylated lysozyme.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

effect of mutation on chemical shifts66
Effect of mutation on chemical shifts

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

effect of mutation on relaxation rates67
Effect of mutation on relaxation rates

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

model for unfolded ensemble
Model for unfolded ensemble

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

compactness by nmr
Compactness by NMR

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum70
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

nmr spectroscopy71
NMR spectroscopy
  • Size
  • Stability
  • Sample homogeneity
  • Need for labeling
  • Quantities and source of biomolecules

General limitations

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

resolution
Resolution

Wide versus small chemical shift dispersion

folded

unfolded

Unfolded proteins have a small chemical shift dispersion.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

1d 1h nmr spectra
1d 1H NMR spectra

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

hsqc spectra
HSQC spectra

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide75

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: S24–S40

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide76

Example Solution NMR of Rhodopsin

It’s a headache.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide77

What is signal 1?

How can you test your hypothesis?

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide78

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:

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide79

Traditional Solution NMR Approaches

Problems with full-length membrane proteins in detergents

  • Size – is not the only problem (Trosy does not work for helical membrane proteins)
  • Conformational exchange – fluctuations in the detergent micelle environment lead to fast relaxation thus signal decay
  • Spin diffusion – cannot deuterate samples from mammalian cells

Problem: Traditional assignment strategies using triple resonance experiments (13C,15N,1H) don’t work

Klein-Seetharaman et al. (2004) PNAS 101, 3409-13.

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide80

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide81

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]

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

you might also want to develop your own biophysical approaches
You might also want to develop your own biophysical approaches…

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide83

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

SH

CF

2

3

Exchange

(TET)

Rho

CF

CH

S

S

3

2

Large-scale expression system for rhodopsin: HEK293 cells

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide84

19F NMR Spectroscopy

Qualitative Changes

dark

light

Dark

Light (3')

23'

43'

63'

10.5

10.0

9.5

9.0

8.5

Chemical shift (ppm relative to TFA)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide85

Proximity

19F-19F Nuclear Overhauser Effect

CF

CH

S

S

3

2

CF

CH

S

S

3

2

Rho

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

magic angle spinning
Magic angle spinning

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

enhancing resolution in solid state nmr
Enhancing resolution in solid state NMR

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

Aligned:

Magic angle spinning:

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

current status of solid state nmr
Current Status of solid state NMR

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

redor
REDOR

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum90
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)

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

the components of an nmr spectrometer
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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide92

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

slide93

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

the shim system
The Shim System

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

monitoring shimming
Monitoring Shimming

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

monitoring shimming96
Monitoring shimming

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

objectives of this lecture and practicum97
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!!

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

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

Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture