Solution Protein NMR

Rieko Ishima Dept. of Structural Biology

School of Medicine

Nuclear Magnetic Resonance (NMR)

Nuclear Magnetic Resonance (NMR) spectroscopy

NMR spectroscopy - Resonance

B0

N

S

1H nuclear spin

Hydrogen atom

α

β

NMR spectroscopy - Resonance

α

β

Chemical shift (ppm) ηω

Nuclear spin

B0: Magnetic field strength

α

β hωΗ

(ppm)

At 14 Tesla Magnet Proton resonance frequency (ωH) = 600 MHz Carbon resonance frequency (ωC) = 150 MHz Nitrogen resonance frequency (ωH) = 61 MHz

NMR Spectroscopy - Resonance

Correlation

H H-N

N

ppm H

Observation of N-H chemical shifts

H C

CH H3C CH3

C O N H

C H

CH3 O C N

H

H C

CH NH2

C O

OC

H N

N - H

1H (ppm)

15N

(ppm

)

An N-H correlation spectrum

1H (ppm)

15N

(ppm

)

1H

15N

ωN

ωH

Three-dimensional NMR spectra

1H

13C

1H

15N

13C

Three-dimensional NMR spectra

1H

13C

1H

15N

13C

Backbone signal assignments CBCACONH

HNCA

Cαi+1

1H (ppm)

15N (ppm

)

T96

1H-15N NMR correlation spectra of HIV-1 protease Once signals are assigned,

the information is used to identify the sides that are affected by a mutation.

1H (ppm)

Dimer (PR) Monomer (PRR87K)

Ishima et al (2001) J Biol Chem. 276, 49110-6.