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

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NMR SpectroscopyQuestion and Answer Session

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

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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

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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

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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

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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

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Homework Assignments
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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

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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

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• click on Topspin icon and open the program

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

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• Right click on MB3_Jan2007

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• Select Expand Show PULPROG/Title

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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

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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

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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.

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

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• 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

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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.

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Phasing
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Sample A. 20-23
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(No Transcript)
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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
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Sample B. 1-7, 60-61, 110-113
3 hsqc
8 selabs
4 zg
2 zgpr
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Sample D. 30-33
33 hsqc
32 selabs
31 zgpr
30 zg
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Sample E. 40-43
42 selabs
41 zgpr
40 zg
43 hsqc
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Sample F. 10-15, 70-71, 100-103
103 hsqc
102 selabs
101 zgpr
100 zg
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Sample H. 50-53
52 selabs disregard this spectrum
53 hsqc
51 zgpr
50 zg
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Parameters to watch out for

• ns
• rg
• will affect actual intensity measured.

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Several different assignment strategies exist
Most easily automated

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

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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

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This is a cluster
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HNCO
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HNCA
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HNCACO
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HNCOCACB, similar CBCA(CO)NH
Not in HNCOCACB experiment
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HNCOCA
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HNCACB
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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

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

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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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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
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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