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
NMR demo programs … FREE!
http://bmrl.med.uiuc.edu:8080/edusoft.html list os available NMR programs
Acquiring the FID
z
y
x
w
Receiver/transmitter
voltage
time
The receiver coil picks up the signal from the sample.
An analog-to-digital converter “reads” the voltage and
sends it to the computer for data storage.
One Dimensional NMR
z
Mo
y
x
x
x
w
x
90x
FID
FT
time
frequency in ppm
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
COSY 2D Experiment
y
w
x
90x
w
90y
t1
preparation
x
evolution
t2
acquisition
The two dimensions are t1 and t2.
x
COSY 2D Experiment
t1 = 750ms
FT
t1 = 600ms
FT
t1 = 450ms
FT
t1 = 300ms
FT
t1 = 150ms
t1
FT
t1 = 0ms
FT
t2
Typically there will be ~128-to-512
t1 increments in a single 2D data file.
f2


 
t1


The Interferogram
Interferogram
FID
t1
t1
f2
Contour plot.
2D plot of data
f1
f1
Bax and Morris, Jl. Magn. Res.,
42, 501-05 (1981).
f2
f2
NOESY 2D Experiment
90n
90n
t1
preparation evolution
90y
Tm
mixing
t2
acquisition
• 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.
Polypeptide Spin System
7-10 ppm
3.5-6.0 ppm
Denotes spin systems in the individual residues
Denotes the aH-NH COSY connectivities
Denotes the sequential NOE connectivities
“NMR of Proteins and Nucleic Acids” Wuthrich, p131, (1986).
Sequential and Medium Range Distances
“NMR of Proteins and Nucleic Acids” Wuthrich, p118, (1986).
Nonsequential 1H-1H Distances in Proteins
Side Chain Coupling Patterns
diagonal peaks
COSY peaks
+, * relayed COSY
“NMR of Proteins and Nucleic Acids”
Wuthrich, p136, (1986).
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 residues
1286 atoms
1305 bonds
Brookhaven 1A3S
4 alpha helical regions
1 or 2 b sheet residues.
Sample NMR Spectra of Ubiquitin
obtained from Georgetown's 500 MHz Unity INOVA NMR Spectrometer
Samples 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 Ubiquitin
obtained from Georgetown's 500 MHz Unity INOVA NMR Spectrometer
Samples courtesy of Ms. Tao Wang (Prof. David Yang's research group)
Sample NMR Spectra of Ubiquitin
obtained from Georgetown's 500 MHz Unity INOVA NMR Spectrometer
Samples courtesy of Ms. Tao Wang (Prof. David Yang's research group)