Structure Determination by NMR

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)