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Protein in solution
Radio waves
1H 800 MHz13C 200 MHz15N 80 MHz
Spectra
Principles of NMR Protein Spectroscopy
Bo 18.79 T
Frequencies and Spectra
Principles of NMR Protein Spectroscopy
How would the 1H NMR spectrum appear?
H
HH
H
H
NH
O
O
HN
H
H
CH3
HH
Ala Phe
10 9 8 7 6 5 4 3 2 1 0 ppm
Principles of NMR Protein Spectroscopy
- NMR is very sensitive to environment
H
HH
H
H
NH
O
O
HN
H
H
CH3
HH
Ala Phe
electronic, bonding
small differences in for each nucleus in molecule
B0
2(Hz)
s = B0
2
ActualGeneral = B0
2(Hz)
is shielding constantcan or of nucleus
-same for 13C, 15N
Chemical Shift
Principles of NMR Protein Spectroscopy
B0
2(Hz) - resonance condition (field + local)
s = B0
2
Chemical Shift
B=Bo(1-) = B0
2recall
=d +p +m +r +e +s
d = e2
3me
re dr0
8
Principles of NMR Protein Spectroscopy
each nucleus has unique chemical shift
H
HH
H
H
NH
O
O
HN
H
H
CH3
HH
Ala Phe
10 9 8 7 6 5 4 3 2 1 0 ppm
similar for 13C, 15N
1H spectrum
Principles of NMR Protein Spectroscopy
Chemical Shift
4.80 ppm
= 106 sample - ref ppmref
ref = internal standard (0 ppm)
1H @ 14.09T sample= 599.2933272 MHzDSS DSS = 599.2904502 MHz
= 4.80 ppmFactors out B0 - is same at any B
DSS - dimethyl-silapentane-sulphonate
2877
Principles of NMR Protein Spectroscopy
Chemical Shift
DSS 1H @ 14.09T, DSS= 599.2904502 MHz
010
070
13C @ 14.09T ref = 13C * DSS 1H
=150.6913020
Chemical Shifts of Proteins
1H spectrum
In generalnuclei found in “ranges” of chemical shift
10 9 8 7 6 5 4 3 2 1 0 ppm
NH
Aromatics
HH
CH3
100 90 80 70 60 50 40 30 20 10 0 ppm
C C CH3
13C spectrum C
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 differentdependence on placement in sequence and secondary structure
Example
H
HH
H
H
NH
O
O
HN
H
H
CH3
HH
Ala Phe
8.19
7.03-7.11
4.61
4.261.37 8.39
2.97, 3.00
Principles of NMR Protein Spectroscopy
Spin-spin Coupling
Principles of NMR Protein Spectroscopy
A ABAB
= B0
2B0
2+J
J
Spin-spin Coupling
Principles of NMR Protein Spectroscopy
1H(15N)
1H-1H 0-20 Hz1H-13C 125-160 Hz1H-15N ~ 93 Hz
2nI +1 lines
13C(1H)
93 Hz 143 Hz
-CH3-NH
Spin-spin Coupling
Principles of NMR Protein Spectroscopy
1H-1H 0-20 Hz
H
HH
H
H
NH
O
O
HN
H
H
CH3
HH
3J
3J = 6.98cos2 - 1.38cos +1.72
= - 60˚
Other Coupling
Principles of NMR Protein Spectroscopy
D = || (3cos2 - 1)
Dipolar Interaction
2
|| = - 12h
42r3
Bo r12
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