LECTURE 3
Coherence
depicted as distributions molecules with jl of 1H closest to —y selected
decomposed distributions based on # of 13C/15N being closer to -\-z vs. to —z in the selected molecules
depicted as arrows     decomposed arrows
In-phase
Anti-phase
MfJL
7 * f I I *
iter'
In-phase:
i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_i_
0    1    2    3    4    5    6    7    8    9   10  11   12 13  14  15  16 17  18  19 20 21   22 23 24
I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_
0    1    2    3    4    5    6    7    8    9   10  11   12 13  14  15  16 17  18  19 20 21   22 23 24
_I_I_I_I_I_ \\-^_I_I_I_I_I_I_I_I_I_I_I_I _I_I_I_I_I_
0    1    2    3    4    5    6    7    8    9   10  11   12 13  14  15  16 17  18  19 20 21   22 23 24
x
Free coherence evolution
Refocusing spin echo
Decoupling spin echo
13C or15N -
1
Simultaneous spin echo
INEPT
1
H
13C or15N
1
1
H
3C or15N
4J | 4J
I
INEPT
1
H
13C or15N
1
1
H
3C or15N
1
i^j i
INEPT
1
1
H
3C or15N
1
3C or15N
INEPT
1
H
13C or15N
1
1
H
3C or15N
J_ 4J
1
| 43
I
INEPT
INEPT vs. direct excitation
1H
13C or15N
HSQC Spectroscopy
±   ± y
| 4J | 4J |
X
13C or15N-1 \
Multiple quantum coherences
Multiple quantum coherences
Multiple quantum coherences
Multiple quantum coherences
Decoupling in direct dimension
C22
2	ttJ
	
Decoupling in direct dimension
•■Hi
1
H
1
3C or15N
| 4J | 4J |
x/y
ti
1
4U
J_
4J
Decoupling in direct dimension
[ 2	ttJ
' wow	
1 ^2
1 ^2
N HSQC spectrum of a 20 kDa protein
Benefits of HSQC
• High sensitivity for 1JC or 1DN
(higher by (7i/72)5//2 than by the direct detection
• High resolution
Second dimension and less peaks in spectrum
(only 13C/15N-bonded protons and protonated 13C/15N visible)
• Important structural information 1H-13C and 1H-15N correlation
(it tells us which proton is attached to which 13C or 15N).