J‐coupling,
H‐bonds,
and
Chemical
Shi7
Mapping
Looking
at
protein
structures
•  How
to
find
a
PDB
file
(www.pdb.org)
•  How
to
look
at
a
structure
•  Remember,
structures
are
only
a
model
fit
to
experimental
data!!!
NtrC
–
crystal
structure
bound
to
pepQde
ligand
and
apo
NMR
structure
1F4V
1KRW
J‐coupling
•  Energy
levels
of
nucleus
I
is
affected
by
the
spin
state
of
the
directly
bonded
nucleus
S.
n
J ab N=#
bonds
between
nuclei
a
and
b
H=2πJI•S
•  Directly
bonded
nuclei
share
electrons
€
–  Electrons
are
shared
between
covalently
bonded
nuclei.
–  Spin
state
of
I
polarizes
the
bonded
electrons,
affecQng
the
energy
levels
of
the
S
nucleus
that
shares
those
electrons.
–  J‐coupling
is
due
to
hyperfine
interacQon,
wave
funcQon
must
include
some
s‐orbital
character.
–  Useful
for
assignment,
since
direct
bonding
required
I
Polarized
e‐
S
Assuming
J>0,
γ>0
Slightly
Slightly
higher
lower
energy
energy
I
Polarized
e‐
S
J‐coupling
ββ
ββ
ωI
ωS
αβ
βα
ωI+πJ/2
ωS+πJ/2
αβ
βα
ωI
ωS
αα
ωI‐πJ/2
ωS‐πJ/2
αα
Both
spectral
lines
split
by
πJ
ωS
ωI
ωS
ωI
J‐coupling
•
Magnitude
of
spli_ng
(JAB)
is
proporQonal
to
γAγB.
–  Same
nJAB
measured,
whether
you
measure
A
spectrum
split
by
B
or
B
spectrum
split
by
A.
–  J
is
independent
of
magneQc
field.
•
•
•
Scalar
coupling
strength
depends
on
n,
greatly
aaenuated
for
2‐bond
versus
1‐
bond.
Typical
J‐coupling
values
used
in
protein
NMR
Transfer
via
J‐coupling
is
faster
&
more
efficient
for
stronger
J‐coupling
(1/J).
–  Faster
transfer,
less
relaxaQon
loss.
HN
or
HC
HSQC
very
intense
–  HNCO
beaer
signal
than
HNCA
–  Backbone
transfers
needed
for
assignment
problemaQc
in
large
proteins
with
fast
relaxaQon.
i‐1
One
bond
scalar
couplings
(Hz)
2‐3
bond
scalar
couplings
(Hz)
i
i+1
2J
NCa,
O
H
3JHNCH,
O
H
1 O
1J ,
JC’Ca,
NC’ 1‐10
4‐9
N
7‐11
C
15
N
Cα C
N
55
C
Cα
Cα1J ,
1J ,
CaCb
CH 140
35
Hα R
H C
H C
H
1J ,
1
NH 92
JNCa,
α
β
C γ
3J α ,
HCCH
2‐14
H
β
β
Hydrogen
Bonding
•
J
coupling
can
be
detected
across
a
hydrogen
bond
–
–
–
–
Small
value
(≤1Hz)
Measured
between
15N
and
13C’
since
O
is
not
a
spin
½
nucleus.
Measurable
for
H‐bonds
shorter
than
about
3.3Å
N‐O
distance.
J‐coupling
only
occurs
when
electrons
are
shared.
Therefore,
H‐bonding
must
have
some
covalent
character!!!!
Sheets
tend
to
have
shorter,
stronger
H‐
bonds
than
helices
Cordier
&
Grzesiek
(1999)
JACS
121:1601
Chemical
Shi7
Mapping
‐
ArresQn
•  ArresQn
binds
phosphorylated
GPCRs
•  Map
binding
site
for
IP6
and
heparin,
two
anionic
compounds
that
are
believed
to
bind
in
the
same
locaQon
•  Need
assigned
HSQC,
then
look
at
which
peaks
shi7
upon
addiQon
of
the
compound
–  No
shi7s:
no
direct
interacQon
–  A
few
peaks
shi7:
idenQficaQon
of
specific
binding
site
–  Lots
of
minor
shi7s:
nonspecific
binding
or…
–  Lots
of
peaks
shi7:
large
scale
conformaQonal
change,
allosteric
effects
Zhuang
et
al
(2010)
Biochemistry
41:10473
Chemical
Shi7
Mapping‐ArresQn
IP6
(le7)
and
heparin
(right)
bind
in
the
same
locaQon
in
the
N‐
terminal
region
of
arresQn
and
cause
it
to
release
the
C‐
terminal
tail.
Zhuang
et
al
(2010)
Biochemistry
41:10473
Chemical
Shi7
Mapping
‐
Adk
103
HSQC
spectrum
of
adenylate
kinase
from
Aquifex
aeolicus
at
20
°C,
pH
7
123
104
130
76
10
105
106
180
86
25
101 5642
46
205
203
144
43
Adenlate
kinase
107
181
108
71
106
202
109
110
111
131
139
122
82
148
115
156
77
134
3
150
113
24
114
147 103
115
52 179
116
23
45 168
121
41
118 159
39
97
50
104
109
125
80
110
112
169
196
18
153 55
70
100
199
5126
129
138176
34
174
22
37 53
36 201 15
65
16
160
20089 197
192
69
161
48
154
64 193
88
102
120
54
99
126
173
167
94 57
124
27
83
91 2040 178
68 164
11
135 21 136 165
145
19
204 163 195
116
9695132 175
67
191
158
58
49
17
33 172
47
117
166
133 38
157
66
4
162
93
87
61 10798
92
35 194
151
149
59
177
112
3078
127
63
79 184
206
81186
90
182
114
111
6
2
85
62
188
137
5
183
152
117
118
119
120
185
28
108
122
123
124
125
126
170
72
127
128
129
146
130
131
132
11.0
10.5
10.0
9.5
9.0
8.5
F2 ppm
8.0
7.5
7.0
p
p
m
121
189
105
187
29
F
1
6.5
AMP
+
ATP

2
ADP
Reversible
reacQon,
equilbrium
near
1,
requires
Mg2+
Chemical
shi7
mapping
‐
Adk
Apo
versus
Mg•ADP
(catalysis)
105.0
110.0
F1
115.0
120.0
125.0
130.0
135.0
11.0
10.0
9.0
F2
8.0
7.0
6.0
Large
scale
conformaQonal
change
upon
nucleoQde
binding,
many
residues
shi7
significantly
Chemical
shi7
mapping
‐
Adk
Mg•Ap5A
(inhibited)
versus
Mg•ADP
(catalysis)
105.0
110.0
F1
115.0
120.0
125.0
130.0
135.0
11.0
10.0
9.0
F2
8.0
7.0
6.0
Chemical
shi7
mapping
‐
Adk
AMP
ATP
Aden
&
Wolf‐Watz
(2007)
JACS
129:14003
ADP
Chemical
Shi7
Mapping
Cytochrome
c552
with
and
without
the
CuA
domain
of
cytochrome
c
oxidase.
Transient
interacQon
–
does
not
form
a
long‐lived
complex.
However,
some
small
shi7s
are
sQll
detectable
(fast
on‐off
rate,
so
populaQon‐weighted
average
chemical
shi7
posiQon)
and
can
be
used
to
map
the
binding
interface.
Wienk
et
al
(2003)
Biochemistry
42:6005