Structure Determination by NMR

CHY 431 Biological Chemistry

Karl D. Bishop, Ph.D.

One Dimensional NMR

Two Dimensional NMR

Resonance Assignment Procedures

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

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 and
sends it to the computer for data storage.

z

y

time

voltage

One Dimensional NMR

x

M

o

90

x

y

z

x

x

x



FT

frequency in ppm

time

FID

Two Dimensional NMR

The two principle types of 2D NMR experiments are NOESY and COSY.

These can be either homonuclear,

1

H-to-

1

H, or heteronuclear,

13

C-to-

1

H.

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 t

1

.

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

90

x

90

y

preparation evolution acquisition

t

2

t

1

COSY 2D Experiment

The two dimensions are t1 and t2.

x



y

x

x



COSY 2D Experiment

FT

FT

FT

FT

FT

FT

t

1

= 150s

t

1

= 300s

t

1

= 450s

t

1

= 600s

t

1

= 750s

t

1

= 0s

Typically there will be ~128-to-512
t

1

increments in a single 2D data file.

t

2

f

2

t

1





 





t

1

The Interferogram

f

2

f

1

f

2

f

1

t

1

f

2

t

1

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.
• t

2

is the amount of time to acquire each

FID.
• t

1

is an incremented time period or

evolution time.
• T

m

is the “mixing time” during which the

dipolar through-space coupling is allowed.

90

n

90

y



m

t

2

t

1

90

n

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 sequentialconnectivities

7-10 ppm

3.5-6.0 ppm

Sequential and Medium Range Distances

“NMR of Proteins and Nucleic Acids” Wuthrich, p118, (1986).

Nonsequential

1

H-

1

H 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 residues
1286 atoms
1305 bonds
Brookhaven 1A3S
4 alpha helical regions
1 or 2  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)