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NMR%20Spectroscopy%20Question%20and%20Answer%20Session

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NMR Spectroscopy Question and Answer Session Judith Klein-Seetharaman Department of Structural Biology jks33_at_pitt.edu – PowerPoint PPT presentation

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Title: NMR%20Spectroscopy%20Question%20and%20Answer%20Session


1
NMR SpectroscopyQuestion and Answer Session
  • Judith Klein-SeetharamanDepartment of Structural
    Biology
  • jks33_at_pitt.edu

2
What did we do?
  • NMR instrumentation
  • NMR sample preparation considerations
  • Water signals
  • Water suppression
  • Detergents
  • NMR setup downstairs
  • zg
  • zgpr
  • selabs
  • hsqc
  • Spectral processing Topspin Software

3
What did we do then?
  • Converting raw spectral formats to other formats
    using NMRpipe
  • Example HIV protease relaxation data
  • Assignment of signals with one strategy
  • HNCO
  • HNCOCACBG
  • HNCOCA
  • HNCACB
  • HNCA
  • HNCACO
  • Example Ubiquitin assignment with NMRviewJ

4
What would be the next step?
  • For determining a structure with NMR
    spectroscopy, after assignment, we would
  • Collect constraints
  • NOE
  • Dipolar coupling
  • Scalar coupling constants (gives dihedral angles)
  • Solvent exchange
  • Calculate a structure based on these constraints

5
What are other things one can do with NMR?
  • For study of dynamics
  • Measure and analyze T1, T2, HetNOE data
  • We went through T1 analysis in computer lab
    (conversion of raw data with NMRpipe, analysis
    with NMRviewJ)
  • T2 was your homework
  • Other studies might include chemical shift
    perturbations
  • Temperature titrations

6
Homework Assignments
7
Homework Part 1
  • NMR instrumentation
  • NMR sample preparation considerations
  • Water signals
  • Water suppression
  • Detergents
  • NMR setup downstairs
  • zg
  • zgpr
  • selabs
  • hsqc
  • Spectral processing Topspin Software

8
Experiments Run
  • zg records all 1H's in your sample
  • zgpr same as zg but with water suppression
  • selabs the way we set it up, it records only
    NH region
  • hsqcfpgpf3gphwg in short hsqc records all
    1H's attached to 15N

9
  • click on Topspin icon and open the program

10
  • click on guest, expand MB3_Jan2007 directory
  • This is the directory that contains all of the
    data that was acquired in class.

11
  • Right click on MB3_Jan2007

12
  • Select Expand Show PULPROG/Title

13
Samples
  • Sample IDs Experiment IDs
  • B 1-7
  • F 10-15
  • A 20-23
  • D 30-33
  • E 40-43
  • H 50-53
  • B 60, 61
  • F 70, 71
  • F 100-103
  • B 110-113

14
Samples
  • 40 zg of sample E
  • 41 zgpr of sample E
  • 42 selabs of Sample E
  • 43 hsqc of Sample E
  • 50 zg of Sample H
  • 51 zgpr of Sample H
  • 52 selabs of sample H
  • 53 hsqc of sample H
  • 60 zg of sample B
  • 61 zgpr of sample B
  • 70 zg of sample F
  • 71 zgpr of sample F
  • -----------------experiments done with comp bio
    students
  • 100 zg of sample F
  • 1 zgpr of Sample B
  • 2 zgpr of Sample B
  • 3 hsqc of Sample B
  • 4 zg of Sample B
  • 5 zg of Sample B
  • 6 selabs of Sample B
  • 7 selabs of Sample B
  • 10 zg of Sample F
  • 11 selabs of Sample F
  • 12 zgpr of Sample F
  • 13 hsqc of sample F
  • 14 hsqc of sample F
  • 15 hsqc of sample F
  • ----- experiments done with MB3 students
  • 20 zgpr of Sample A
  • 21 zg of sample A
  • 22 selabs of sample A

15
Solving the Mystery of the Samples
  • Drag the experiment that you want to analyze into
    the main purple window area.
  • If you do not see a spectrum, you need to type
    efp
  • This will process the data so that you can see
    the spectrum.
  • In the case of the hsqc it will also ask you for
    an experiment number where to store the processed
    data, you can use the default or enter a number,
  • e.g. 2. Another alternative is to type rser 1
    that will retrieve the first slice of the 2d hsqc
    experiment also.

16
  • To see what acquisition parameters were chosen,
    click on the AcquPars tab, you will see a window

17
  • Notice that you can scroll down to see many
    fields under this tab.
  • If you type ased only those parameters relevant
    to the specific experiment are shown

18
Some tips
  • If you want to move more quickly from experiment
    to experiment, type re and then the experiment
    number, e.g.
  • re 21 gives the experiment stored in directory 21
  • To save a picture of a spectrum select File,
    Export and then give it a file name extension
    indicating which format you would like (e.g.
    .jpg)
  • Note that you may have to phase your spectrum.

19
Phasing
20
Sample A. 20-23
21
(No Transcript)
22
Overlay Sample A
  • From these spectra, we can conclude that we do
    not have 15N label, it i a water sample. It does
    not have detergent in it, or the detergent is
    deuterated.

22 selabs
21 zg
20 zgpr
23 hsqc
23
Sample B. 1-7, 60-61, 110-113
3 hsqc
8 selabs
4 zg
2 zgpr
24
Sample D. 30-33
33 hsqc
32 selabs
31 zgpr
30 zg
25
Sample E. 40-43
42 selabs
41 zgpr
40 zg
43 hsqc
26
Sample F. 10-15, 70-71, 100-103
103 hsqc
102 selabs
101 zgpr
100 zg
27
Sample H. 50-53
52 selabs disregard this spectrum
53 hsqc
51 zgpr
50 zg
28
Parameters to watch out for
  • ns
  • rg
  • will affect actual intensity measured.

29
Several different assignment strategies exist
Most easily automated
  • HNCO
  • HNCOCACB
  • HNCOCA
  • HNCACB
  • HNCA
  • HNCACO

30
Homework Part 2
  • Converting raw spectral formats to other formats
    using NMRpipe
  • Example HIV protease relaxation data
  • Assignment of signals with one strategy
  • HNCO
  • HNCOCACBG
  • HNCOCA
  • HNCACB
  • HNCA
  • HNCACO
  • Example Ubiquitin assignment with NMRviewJ

31
This is a cluster
32
HNCO
33
HNCA
34
HNCACO
35
HNCOCACB, similar CBCA(CO)NH
Not in HNCOCACB experiment
36
HNCOCA
37
HNCACB
38
What are other things one can do with NMR?
  • For study of dynamics
  • Measure and analyze T1, T2, HetNOE data
  • We went through T1 analysis in computer lab
    (conversion of raw data with NMRpipe, analysis
    with NMRviewJ)
  • T2 was your homework
  • Other studies might include chemical shift
    perturbations
  • Temperature titrations

39
Dynamics in folded/unfolded lysozyme
Unfolded
Folded
Smaller rates more flexible
40
T1 for HIV protease
41
T2 for HIV protease
42
T2 for HIV protease
43
  • From last class in case there are questions

44
NMR parameters
Chemical Shift
H2O
methyl
aromatic
Trp-side-chain NH
OH
aliphatic
Backbone NH
Side-chain HN
Ha
Spectrum see handout
45
NMR of membrane proteins
In detergent micelle
In lipid bilayer
http//www.elmhurst.edu/chm/vchembook/558micelle.
html
46
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
47
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
48
2d HSQC
49
1d projection of HSQC
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