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Structure Determination by NMR. CHY 431 Biological Chemistry Karl D. Bishop, Ph.D. One Dimensional NMR Two Dimensional NMR Resonance Assignment Procedures. http://www.cm.utexas.edu/hoffman/index_gang.html. http://www.chem.vt.edu/chem-dept/hbell/simulation/VTNMR.html - PowerPoint PPT Presentation
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Structure Determination by NMR
CHY 431 Biological ChemistryKarl D. Bishop, Ph.D.
One Dimensional NMR
Two Dimensional NMR
Resonance Assignment Procedures
http://www.chem.vt.edu/chem-dept/hbell/simulation/VTNMR.htmlNMR demo programs … FREE!http://bmrl.med.uiuc.edu:8080/edusoft.html list os available NMR programs
http://www.cm.utexas.edu/hoffman/index_gang.html
Acquiring the FID
x
Receiver/transmitter
The receiver coil picks up the signal from the sample.An analog-to-digital converter “reads” the voltage andsends it to the computer for data storage.
z
y
timevoltage
One Dimensional NMR
x
Mo
90x
y
z
xx x
FT
frequency in ppmtime
FID
Two Dimensional NMR
The two principle types of 2D NMR experiments are NOESY and COSY.
These can be either homonuclear, 1H-to-1H, or heteronuclear, 13C-to-1H.
A 2D data set can be thought of as a stack of 1D files.
Each 1D file is different from the next by a change in t1.
All other parameters are kept constant except the phase of the pulses.
Fourier transformation of each 1D in the t2 domain creates an interferogram.
The t1 domain is then Fourier transformed resulting in a 2D file with the frequency in each dimension.
This 2D file will provide a map of all spin-to-spin correlations
90x 90y
preparation evolution acquisition
t2t1
COSY 2D Experiment
The two dimensions are t1 and t2.
x y
xx
COSY 2D Experiment
FT
FT
FT
FT
FT
FT
t1 = 150s
t1 = 300s
t1 = 450s
t1 = 600s
t1 = 750s
t1 = 0s
Typically there will be ~128-to-512 t1 increments in a single 2D data file.
t2 f2
t1
t1
The Interferogram
f2
f1
f2
f1
t1
f2
t1
Interferogram FID
2D plot of data
Contour plot.
Bax and Morris, Jl. Magn. Res., 42, 501-05 (1981).
NOESY 2D Experiment
• The two dimensions are t1 and t2.• t2 is the amount of time to acquire each FID.• t1 is an incremented time period or evolution time.• Tm is the “mixing time” during which the dipolar through-space coupling is allowed.
90n 90y
m t2t1
90n
preparation evolution mixing acquisition
Polypeptide Spin System
“NMR of Proteins and Nucleic Acids” Wuthrich, p131, (1986).
Denotes spin systems in the individual residues
Denotes the H-NH COSY connectivities
Denotes the sequentialconnectivities
7-10 ppm
3.5-6.0 ppm
Sequential and Medium Range Distances
“NMR of Proteins and Nucleic Acids” Wuthrich, p118, (1986).
Nonsequential 1H-1H Distances in Proteins
Side Chain Coupling Patterns
“NMR of Proteins and Nucleic Acids” Wuthrich, p136, (1986).
diagonal peaks
COSY peaks
relayed COSY+, *
Side Chain Coupling Patterns
“NMR of Proteins and Nucleic Acids” Wuthrich, p136, (1986).
Backbone Coupling in Peptides
“NMR of Proteins and Nucleic Acids” Wuthrich, p119, (1986).
NMR Analysis of Ubiquitin
158 residues1286 atoms1305 bondsBrookhaven 1A3S4 alpha helical regions1 or 2 sheet residues.
Sample NMR Spectra of Ubiquitinobtained from Georgetown's 500 MHz Unity INOVA NMR SpectrometerSamples courtesy of Ms. Tao Wang (Prof. David Yang's research group)
2D COSY of Ubiquitin
Cavanaugh et al., 1996
NMR Analysis of Ubiquitin
Cavanaugh et al., 1996
Sample NMR Spectra of Ubiquitinobtained from Georgetown's 500 MHz Unity INOVA NMR SpectrometerSamples courtesy of Ms. Tao Wang (Prof. David Yang's research group)
Sample NMR Spectra of Ubiquitinobtained from Georgetown's 500 MHz Unity INOVA NMR SpectrometerSamples courtesy of Ms. Tao Wang (Prof. David Yang's research group)