Principles of NMR Protein Spectroscopy

Frequencies and Spectra

Spectra
Radio waves
 Principles of NMR Protein Spectroscopy

How would the 1H NMR spectrum appear?

Hζ
Hε Hε

Hδ Hδ

O Hβ
H Hα Hβ
N
N
Hα CH3 H O
Ala Phe

10 9 8 7 6 5 4 3 2 1 0 ppm
 Principles of NMR Protein Spectroscopy

Chemical Shift
Hζ
- NMR is very sensitive to environment Hε Hε

electronic, bonding Hδ Hδ

O Hβ
H Hα Hβ
N
N
Hα CH3 H O
Ala Phe
small differences in ν for each nucleus in molecule
 Principles of NMR Protein Spectroscopy

Chemical Shift

B=Bo(1-σ) recall ν = γB0

2π
νs = γB0 −σ γB0 (Hz) - resonance condition (field + local)
2π 2π

σ = σd + σp + σm + σr + σe + σs
8

σd = µοe2 rρe dr
3me 0
 Principles of NMR Protein Spectroscopy
Hζ
each nucleus has unique chemical shift Hε Hε

similar for 13C, 15N Hδ Hδ

O Hβ
H Hα
1H Hβ
spectrum N
N
Hα CH3 H O
Ala Phe

10 9 8 7 6 5 4 3 2 1 0 ppm
 Principles of NMR Protein Spectroscopy

Chemical Shift
2877
δ = 106 νsample - νref ppm
νref

νref = internal standard (0 ppm)

4.80 ppm DSS - dimethyl-silapentane-sulphonate
1H @ 14.09T νsample= 599.2933272 MHz
DSS νDSS = 599.2904502 MHz
δ = 4.80 ppm
Factors out B0 - δ is same at any B0
 Principles of NMR Protein Spectroscopy

Chemical Shift

DSS 1H @ 14.09T, νDSS= 599.2904502 MHz

10 0

13C @ 14.09T νref = γ13C * νDSS

γ1H
=150.6913020
70 0
 Principles of NMR Protein Spectroscopy

Chemical Shifts of Proteins

In general
nuclei found in “ranges” of chemical shift
1H spectrum
Aromatics CH3
Hβ,γ,δ
NH Hα
10 9 8 7 6 5 4 3 2 1 0 ppm

13C spectrum Cγ,δ

Cα Cβ CH3

100 90 80 70 60 50 40 30 20 10 0 ppm
 Principles of NMR Protein Spectroscopy

Protein Database

BioMagResBank

http://www.bmrb.wisc.edu/ Reference

Reference Information Chemical Shift Statistics Derived

from the BMRB Database for:
Restricted Set of Protein Chemical Shifts
Full Set of Protein Chemical Shifts
Restricted Set of DNA Chemical Shifts
Full Set of DNA Chemical Shifts
Restricted Set of RNA Chemical Shifts
Full Set of RNA Chemical Shifts
 Principles of NMR Protein Spectroscopy

BioMagResBank

actual chemical shifts for each amino acid are different

dependence on placement in sequence and secondary structure
Hζ
Example Hε Hε
7.03-7.11
Hδ Hδ
8.19
O 4.61 Hα Hβ 2.97, 3.00
H
Hβ
N
N
4.26 Hα CH3 H O
1.37 8.39
Ala Phe
 Principles of NMR Protein Spectroscopy

Spin-spin Coupling
ββ

βα

αβ
αα J
A AB AB
ν = γB0 + γB0σ +J
2π 2π
 Principles of NMR Protein Spectroscopy

Spin-spin Coupling
1H-1H 0-20 Hz
1H-13C 125-160 Hz
1H-15N ~ 93 Hz
2nI +1 lines -NH -CH3
93 Hz 143 Hz

1H(15N) 13C(1H)
 Principles of NMR Protein Spectroscopy

Spin-spin Coupling Hζ
1H-1H 0-20 Hz Hε Hε

Hδ Hδ
3J
O Hβ
H Hα Hβ
N
N
Hα CH3 H O

3J = 6.98cos2θ - 1.38cosθ +1.72

θ = φ - 60˚
 Principles of NMR Protein Spectroscopy

Other Coupling

Dipolar Interaction

νD = ν|| (3cos2θ - 1) ν|| = - γ1γ2h

2 4π2r3

θ
Bo
r12