Lecture
Basics NMR of NMR Nuclear Spin Resonance Frequency Nuclear Shielding Spin Coupling Relaxation Applications of NMR Small Molecules Macromolecules Beast of Biochemistry
Nuclear Magnetic Resonance Spectroscopy
valiant magnet
Basics of NMR
Nuclear Spin Caution: nothing that I tell you is true
Nuclear Spin Resonance Frequency Nuclear Shielding Coupling Relaxation
Resonance Frequency
External Magnetic Field
Resonance Frequency
=
RF
FT
Resonance Frequency
1H
1.2
Resonance Frequency
Nucleus (Natural Abundance)
1H 19F
Gyromagnetic Ratio
1H
1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1
31P 13C
(>99%)
(100%)
(100%)
13C
13C
(1.1%) (<0.01%) 15N (0.4%)
2H
1.2
Sensitivity
Ethanol in a Magnetic Field
...After a 90 1H RF Pulse
13C 13C
13C
13C 13C
13C
16O
16O
Nuclear Shielding
13C
1H
Chemical shift: specific frequency (Hertz (s-1)) at which nuclei resonate.
(shift in Hz (1/s))
ppm =
(spectrometer frequency in MHz)
Spin Coupling
Approximately Equal to T2 in Small Molecules Much Longer than T2 for Large Molecules
Relaxation
T1 Enthalpic Recovery of Z Magnetization T2*
Slow in Small Molecules Fast in Large Molecules 0
1H
12C 16O
12C
Equivalent Energy
Entropic Loss of X,Y Magnetization
0
1
1
2
2
3
3
Time (S.)
The Bottom Line
1) Spin nuclei are useful for NMR 2) Resonance frequency indicates the atom 3) Chemical shift indicates the functional group 4) Coupling indicates molecular connectivity 5) Relaxation reflects the overall environment
Real World NMR Properties
line width height (intensity)
106 Hz
For small molecule 1D 1H spectra of liquids, peak area is directly proportional to the number of 1H atoms in a given environment
splitting (coupling)
1 chemical shift
Natural Products Chemistry
What is this creature?
4 3
Natural Products Chemistry
MES
4 3 2 1 3,4 1,2 1 1,2 3
2
2 1 1
2
4
3,4
C6H12NO4S (from mass spectrometry)
1H-13C
HMBC Shows long range 1H-13C connectivity
35
Metabolomics
In vivo Concentration (mM)
35s Control
30
35s NaCl
Athk1 Control
Athk1 NaCl
WS Control
WS NaCL
Which creatures are in this critter?
25
Monitor Pathway Flux
20
Understand Mutants
15
10
5
Myo-inositol
0
Leucine Succinate
Histidine
Citrulline
Valine
Glycine
Proline
Arginine
4-Aminobutyrate
Asparagine
Glutamate
100
B6 obob BTBR obob B6 +/+ BTBR +/+
50
Healthy
0
Classify Samples
-50
Disease Resistant
-100 0
-100
Diseased
100
200
Glutamine
Threonine
Maltose
Alanine
Malate
Glucose
Serine
Fructose
Sucrose
Lactate
Urea
Magnetic Resonance Imaging
What is in this critter?
T1
NMR Structure Determination
T2*
membrane proteins
multi-domain proteins
RNA 0 1 2 3 partially disordered proteins
Kd ~ M - mM
low-affinity complexes
Sample Requirements
protein size limit
13C, 15N, 2H
Structure Determination
sample preparation data collection
RNA
~ 45-50 nt
13C, 15N case-by-case
~ 30 kDa
13C, 15N large proteins
labeling
2H:
2H:
concentration volume salt D 2O pH stability
0.5 2 mM 250 350 L minimal
0.5 1.5 mM
data processing
resonance assignment conformational constraints structure calculation structure validation
100 mM of a monovalent positive ion 5 10 %
~3 8.5
~ 5.5 8
several days to weeks
Resonance Assignment
properties of an NMR peak:
Resonance H
line Assignment
A56 width height (intensity)
106 Hz
I57 H T5 H
Leu21 H
splitting (coupling)
1 chemical shift
Conformational Constraints
and angles
C, etc. peptide plane
Conformational Constraints
and angles
L11: ~ 126 30 ~ 132 30
TALOS
-helix: = 57 = 47
C -sheet:
= 130 = 123
bad prediction ambiguous good prediction prediction
Conformational Constraints
1H
Conformational Constraints
1H
- 1H Nuclear Overhauser Enhancements (NOEs)
- 1H Nuclear Overhauser Enhancements (NOEs)
H
H
H
H
H H
H H H H H
4.75
H H H H H H
2.5 6
H
H
H
H
H H H
H H H 2.75 H
Conformational Constraints
1H
Conformational Constraints
1H
- 1H Nuclear Overhauser Enhancements (NOEs)
H H H H H H H H H H H H H
3.5
- 1H Nuclear Overhauser Enhancements (NOEs)
H H H H H H H H H H H H H
4.75
H H H H H
H H H H H H H H H H
H H H H
2.75
H
H H
4.75
H
H H H H
3 3.75
H H H H H
H
2.75
H
Conformational Constraints
1H
Conformational Constraints
and angles
1H
- 1H Nuclear Overhauser Enhancements (NOEs)
H H H H
- 1H NOEs
RDCs
H H H H H H H H H H H H H H 2.5 4.5 H H 3.5 H
H
~5
H H N
a
b
H
c
N
(, ) torsion angles information source type of constraint
2 structure
1H
1H NOE
RDC
2, 3 and 4 structure, "bend" peak splitting long range
2, 3 and 4 structure NOESY peak intensity local & long range
13C, 13C
chemical shift
local
etc.
Structure Calculations
primary sequence
Structure Calculations
and angles
vs.
1H
"looseness": lack of conformational constraints (and motion?)
- 1H NOEs ensemble of overlaid structures
RDCs
Structure Determination
sample preparation
Interactions With Ligands
variable
data collection
1-4 weeks 2-3 days + 1-2 months "p" 2-4 months ?
data processing
resonance assignment conformational constraints structure calculation structure validation
1 day
"A"
Interactions With Ligands
molar ratio of
A:p=1:0
Interactions ...