BIOCH 590: Biomacromolecules
89%'91%:(
Spring 2010
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Key References: Tinoco Chapter 10; van Holde Chapter 12;
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Copyright: Jianhan Chen
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“Complex” features of NMR
spectra of chemicals
EFNMR
~10-5 T
~ KHz
$0
1.41 T
60 MHz
11.75 T
500 MHz
$0.5m
L#M(N1&+=&+(O=%+(
18.8 T
800 MHz
21.15 T
900 MHz
>$5m
Y(
J"#=(1+1,&$$<("+3$%&.&+6(#/73$%B16<(L6/(3=<.1#1.6.M(
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" s = " 0 (1 # $ )
H NMR Chart Paper
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!
"s =
relative
intensity
# s $ # ref # s $ # ref
%
#0
# ref
C NMR Chart Paper
low
field
high
field
low
field
high
field
high
frequency
low
frequency
high
frequency
low
frequency
TMS
TMS
–  QB3'%..%4(1+(337(
–  -%I%'%+#%(.6&+4&'4H(6%6'&7%6=<$.1$&+%((L?)JM(L/'(EEJM(
!
13
1
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10
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0
5
! ppm
less shielded
200
0
100
! ppm
more shielded
Nuclei which absorb at higher field are more shielded
from the applied Bo field by their respective Be fields. For
these nuclei (Bo-Be) is smaller and correspondingly a lower
frequency " is required to achieve resonance.
http://orgchem.colorado.edu/hndbksupport/nmrtheory/chemshift.html
[(
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Why do different protons absorb at different frequencies of the B1 field?
?<31#&$(O=%71#&$(J=1>.(
Electronegative elements
such as oxygen pull
electron density away from
the hydrogen nucleus.
•  ]/#&$(.=1%$41+*H(=1*=%'(%$%#6'/+(4%+.16<(@i(7/'%(.=1%$41+*(
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O
C
H
O
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H
This decreases the magnitude
of the shielding Be field and increases
(Bo-Be).
H
methyl acetate
C
H
C
H
O
H
H
methoxy
!=3.7 ppm
methyl
!=2.1 ppm
Circulation of π electrons creates magnetic fields which contribute to the Be field. The contribuiton is a
function of orientation and consequently is an anisotropic effect.
" s = " 0 (1 # $ )
Bo
Bo
Be
R
!
C
H Be adds
to Bo near
the proton
C
R
R
Be
Be adds
H to Bo near
Be
H
C
the proton
Be substracts
from Bo near
the proton
Be
C
R
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Be
Be
http://orgchem.colorado.edu/hndbksupport/nmrtheory/chemshift.html
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R
NH
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R
OH
–  O/73&'%4(6/('&+4/7(#/1$('%I%'%+#%(9&$"%.(
R
R
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Cl
OH
H H 2C
Ph
CH3
R
R
O
O
H
H
R
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CH3
R
H
R
O
R
NR2
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R
O
CH3
CH3
TMS
10
9
8
7
6
5
! ppm
4
3
2
1
0
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http://orgchem.colorado.edu/hndbksupport/nmrtheory/chemshift.html
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Mielke and Krishnan, Prog. NMR Spect (2009) L#M(N1&+=&+(O=%+(
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CH3 CH2 OH
•  ?='/"*=(#=%71#&$(A/+4.(L%$%#6'/+(.31+M(
“Complex” features of NMR spectra of chemicals
(ethanol: CH3 CH2 OH shown)
–  E%#%&.%.(:16=(7/'%(A/+4.H(+/(7/'%(6=&+(I/"'(A/+4.(
–  N(d(T(@(FT(Gn(L4%3%+4.(/+(#=%71#&$(+&6"'%(&+4(.6'"#6"'%M(
–  N(L7%&."'%4(1+(GnM(1.(c%$4(.6'%+*6=(1+4%3%+4%+6f(
•  ]%&4.(6/(.3$1p+*(/I(6=%(7&*+%,#(.31+(%+%'*<($%9%$.;(6=".;(.1*+&$.(
–  K+(6=%($1716(/I(N(qq(#=%71#&$(.=1>(41o%'%+#%(Lc'.6(/'4%'a:%&e(#/"3$1+*M(
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Additional splitting if coupled to multiple
groups of (equivalent) protons
Weak coupling
Strong coupling
AX2Y
http://www-keeler.ch.cam.ac.uk/lectures/index.html
L#M(N1&+=&+(O=%+(
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J&73$%(!)-(J3%#6'&(
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http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm
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–  ?DH(J31+@$&p#%('%$&B&,/+(L$/+*16"41+&$('%$&B&,/+;%%+6=&$31#('%$&B&,/+MH('%#/9%'<(/I(
%`"1$1A'1"7(3/3"$&,/+.(L&+4(6=".(&($/..(/I(.1*+&$(&+4(%+%'*<M(
–  ?FH(J31+@.31+('%$&B&,/+(L6'&+.9%'.%('%$&B&,/+;(%+6'/31#('%$&B&,/+MH($/.6(/I(
x#/=%'%+#%y(L:16=/"6(&($/..(/I(%B#16%4(.6&6%(3/3"$&,/+(/'(%+%'*<M(
–  5(3/:%'I"$(7%&+(6/($/#&$(.6'"#6"'%.(
–  )1+17&$(+%&'([T(6/'.1/+(&+4(
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–  Q731'1#&$(3&'&7%6%'.(Lcp+*M(
–  )"$,3$%(./$",/+.((
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#v = 1/T2
(*: !="-60 for proteins)
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•  -C(.&6"'&,/+(/I(/+%(.31+(#&".%.(
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–  ?"7A$1+*(,7%(d(+.(L(/'(:a(I'%`"%+#<(/I(_GnM(
–  ]&'*%'(7/$%#"$%.(6"7A$%.(.$/:%'(&+4(6=".(7/'%(%o%#,9%(1+(1+4"#1+*(
'%$&B&,/+(L.=/'6%'(?D;(?FM(
–  C&.6%'('%$&B&,/+(&6(=1*=%'(c%$4.(((
The largest component of
the spectral density
function, J($), is obtained
when %c-1 & $0.
•  !8Q(A"1$4"3('&6%d(Da'W(L413/$%@413/$%M(
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•
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N@#/"3$1+*H(&4r&#%+6(3'/6/+.(&+4($/#&$(#/+I/'7&,/+.(
-%$&B&,/+(,7%.H(4<+&71#.(L6"7A$1+*;(1+6%'+&$(&+4(%B#=&+*%M(
!8QH(.=/'6@'&+*%(.3&,&$(41.6&+#%(Lq(W({M(|(L.$/:M(4<+&71#.((
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5$$(6=%.%(`"&+,,%.(#&+(A%(&##"'&6%$<(7%&."'%4;(/>%+;(I/'(&$$(
+"#$%1(1+(6=%(.<.6%7X(?=1.(1.(:=<(!)-(#&+(A%(%B6'%7%$<(3/:%'I"$X(
Additional reading: Dr. James Keeler’s Lectures (U of Cambridge)
http://www-keeler.ch.cam.ac.uk/lectures/index.html
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!)-(6%#=+1`"%.(."#=(&.(3"$.%4(!)-(&+4(7"$,417%+.1/+&$(!)-((
•  ~%#6/'(7/4%$(1.(6=%($&+*"&*%(/I(!)-H(/+$<('1*/'/".(1+(&(I%:(#&.%.;(A"6(
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•  0"$e(7&*+%,n&,/+H(+%6(7&*+%,n&,/+(9%#6/'(&$1*+.(:16=(0T(
•  )/(71*=6(A%(x'/6&6%4y(A<(&('&41/I'%`"%+#<(3"$.%X((
•  8+#%(,$6%4(&:&<(I'/7(6=%(n(&B1.;(6=%(7&*+%,n&,/+(
9%#6/'('/6&6%.(&A/"6(6=%(41'%#,/+(/I(6=%(7&*+%,#(
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•  !)-(%B3%'17%+6.(4%6%#6(6=%(3'%#%..1/+(/I(6=%(
7&*+%,n&,/+(9%#6/';(."#=(&.(A<(3$&#1+*(&(.7&$$(#/1$(
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•  -%$&B&,/+.(&+4(I'%%(1+4"#,/+(4%#&<(LCKEM(
(only x coil shown)
T1
T2
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr2.htm
L#M(N1&+=&+(O=%+(
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•  0'&@e%6(+/6&,/+H(( # " i*Qˆ " j d$ %< i Qˆ j >
–  .31+(.6&6%.H(!i(&+4(#iH((q!!i(RD;(q##i(RD;(&+4(q!#i(RT(
•  J31+(&+*"$&'(7/7%+6"7(/3%'&6/'.H(KB;(K<;(&+4(Kn(L#/''%.3/+4(6/(B;(<;(&+4(n(
!
#/73/+%+6.(/I(&+*"$&'(7/7%76"7M((
H = " 0 Iz + "1 cos(" RF t)Ix
–  J31+(.6&6.(!i(&+4(#i(&'%(%1*%+.6&6%.(/I(KnH(Kn!i(R(S(Ä!i((
–  KB(&+4(K<H((
•  G&71$6/+1&+H( (
%%
%
%
%
(
%
(
%
(lab frame)
(rotating f.)
H = (" 0 # " RF )Iz + "1Ix ~ "1Ix
(R($0(Kn(L.1+*$%(I'%%(.31+M(
%
%
%
%
%(6:/(:%&e$<(#/"3$%4(.31+.M%
= "1I1x + "1I2x + ...
(multiple spins)
•  Å=&6(4/%.(&(3"$.%(4/H(#=&+*%(6=%(41'%#,/+(/I(7&*+%,n&,/+(9%#6/'(
!
# = $1!p.
"3(
(([T(3"$.%H(# R([T/(
D\T(3"$.%H(#(R(D\T/(
LB@3"$.%;(<@3"$.%M(
G&'4(3"$.%H($&'*%($D(L=1*=(3/:%';(
.=/'6(4"'&,/+;($&'*%'(A&+4:146=M(
J/>(3"$.%H(.7&$$($1%
L#M(N1&+=&+(O=%+(
F[(
L#M(N1&+=&+(O=%+(
E%+.16<()&6'1B(?=%/'<H(2'/4"#6(83%'&6/'(
•  -%#&.6(/I(J#='Ç41+*%'É.(%`"&,/+(6/(#/+.14%'(%+.%7A$%(&9%'&*1+*(
•  83%'&6/'.(&.(7&6'1B(L1+(6=%(%1*%+9%#6/'(A&.1.M(
•  E%+.16<(7&6'1BH(
!)-(2'&#,#%(
•  8A.%'9&A$%.(&.(6'&#%(/I(7&6'1BH(
!)-(1.(A%#/71+*(&(x.6&+4&'4y(6//$X(?=%(e%<(1.(
+/:(7/'%(6/("+4%'.6&+4(:=&6(16(1.(#&3&A$%(&+4(
$%..(&A/"6(=/:(/+%(:/"$4(&#6"&$$<(#&''<(/"6(6=%(
L4&6&(&#`"1.1,/+M(%B3%'17%+6X(
•  Q9/$",/+(/I(4%+.16<(7&6'1B(
Additional reading: Dr. James Keeler’s Lectures (U of Cambridge)
http://www-keeler.ch.cam.ac.uk/lectures/index.html
L#M(N1&+=&+(O=%+(
PD(
PT(
)/4%'+(!)-(J3%#6'/7%6%'(
C/"'1%'(?'&+.I/'7(!)-(LC?@!)-M(
•  ?=%(A&.1#(3"$.%(&+4(&#`"1'%(%B3%'17%+6(
“pulse sequence”
RF output RF input
1.  The sample is allowed to come to equilibrium.
2.  RF power is switched on for long enough to rotate
the magnetization through 90' i.e. a 90' pulse is
applied. If the pulse is broad (“powerful”) enough,
all protons in the sample are exited.
3.  After the RF power is switched off we start to
detect the signal which arises from the
magnetization as it rotates in the transverse plane.
4.  The free induction decay contains information
about oscillation of all protons. Fourier transform
analysis will thus produce the whole NMR
spectrum.
L#M(N1&+=&+(O=%+(
Magnetic moments of
excess nuclear spins in the
ground state are represented by
vectors. The vectors precess
about the direction of the Bo field.
PP(
The sum of the individual spinvectors
is represented by a large vector in the
direction of the Bo field.
z
z
This causes the net
magnetization to tip off
the z-axis into the yz-plane
and then precess about the
z-axis. This is equivalent to
ground spin states going to excited
spin states by absorption of energy
from the B1 field.
z
The B1 field is
supplied as a
short pulse along Bo
the x-axis.
Bo
y
Bo
Bo
detector
relaxation
y
y
The change in magnetization
x
as a function of time as the net
magnetization vector in the xy-plane
precesses about the z-axis
is observed along the y-axis as My.
y
B1
x
PV(
z
z
x
B1
y
L#M(N1&+=&+(O=%+(
With time, the change in net magnetization caused by the
B1 pulse decays back to the original state with no net
magnetization in the xy-plane.
x
z
z
z
Mz
Magnetization along the y-axis as a function of time after the B1 pulse.
Bo
My
one cycle
FID
relaxation
eye
y
y
Intensity
Fourier
My
y
Bo
detector
y
x
Transformation
x
x
z
The change in magnetization
as a function of time as the net
magnetization vector in the xy-plane
precesses about the z-axis
is observed along the y-axis as My.
With time, the change in net magnetization caused by the
B1 pulse decays back to the original state with no net
magnetization in the xy-plane.
My
time
Mx
x
magnetization in the xy plane
resolved into Mx and
My components
NMR Spectrum
Time Domain Spectrum
intensity
x
The net magnetization
can be resolved into
Bo
y- and z-axis components.
The component in the xy-plane
precesses about the z-axis.
10
Frequency
Domain Spectrum
5
! ppm
0
http://orgchem.colorado.edu/hndbksupport/nmrtheory/chemshift.html
!)-(?D(-%$&B&,/+(
J31+(Q#=/(
The most famous NMR experiment: the
magnetization ends up along the same
axis, regardless of the values of ! and
the offset, ". This is achieved by using
180 pulse as refocusing pulse.
L#M(N1&+=&+(O=%+(
O/=%'%+#%(
•  56(%`"1$1A'1"7;(L3=&.%M(/I(6'&+.9%'.%(
7&*+%,n&,/+(1.(#/73$%6%$<('&+4/7(
&+4(&9%'&*%.(6/(n%'/;(1X%X;(+/(
#/=%'%+#%X(
•  Ñ3/+(&33$<1+*(&(-C(3"$.%(L.&<;(&([T(
3"$.%(&$/+*(@BM;(6=%(+%6(7&*+%,n&,/+(
&$/+*(n@&B1.(1.(+/:(x'/6&6%4y(6/(&$1*+(
&$/+*(<@&B1.X(?=1.(%B#16&,/+H(DM('%7/9%(
6=%(3/3"$&,/+(41o%'%+#%(/I(6:/(.31+(
.6&6%.(L+/(+%6(7&*+%,n&,/+(&$/+*(nM;(
&+4(FM(%.6&A$1.=%.(&(x#/=%'%+#%yH(6=%(
+"#$%1(+/:(3'/#%..(:16=(6=%(.&7%(
3=&.%(L/+(&+(&9%'&*%(.%+.%MX((
•  ?='/"*=(#/$$1.1/+;(6=%(#/=%'%+#%(1.(
*'&4"&$$<($/.6(LA%I/'%(6=%(%`"1$1A'1"7(
3/3"$&,/+(1.(I"$$<('%@%.6&A$1.=%4M((((
L#M(N1&+=&+(O=%+(
PY(
L#M(N1&+=&+(O=%+(
P\(
?F(-%$&B&,/+()%&."'%7%+6(
z
90 pulse along -x
P[(
http://chem.ch.huji.ac.il/nmr/techniques/other/t1t2/t1t2.html
L#M(N1&+=&+(O=%+(
VT(
2=&.%(O<#$1+*(
DE(!)-(/I(2'/6%1+.(
•  ?/(%$171+&6%("+:&+6%4(.1*+&$.a&',I&#6.(L&+4(.%$%#6(4%.1'%4(#/=%'%+#%M(
•  0<('%3%&,+*(6=%(LDEM(%B3%'17%+6(:16=(&$6%'+&,+*(3"$.%(3=&.%.(&+4(
&9%'&*1+*(6=%(CKE.;(#%'6&1+(.1*+&$.(L&+4(&',I&#6.M(:1$$(#&+#%$X(5+/6=%'(
41'%#6(A%+%c6(1.(%+=&+#%7%+6(/I(.%$%#6%4(.1*+&$@6/@+/1.%('&,/X(((
•  QB&73$%H(MJM((L2=&.%(5$6%'+&,+*(2"$.%(J%`"%+#%M(
methyl
(;1*'
M>?"&'
4&;&)C&/'
D(
Ö(
Ö(
F(
@u(
@u(
P(
@Ö(
@Ö(
V(
u(
u(
amide
~8-9 ppm
aromatic
~7-8 ppm
aliphatic
<4 ppm
These signals add up to
zero! (unless receiver
phase follows pulse
Artifacts that do not
follow the phases will
cancel out!
L#M(N1&+=&+(O=%+(
Multi-dimensional NMR is the work-horse in biomolecular studies.
VD(
L#M(N1&+=&+(O=%+(
VF(
O/''%$&,/+(J3%#6'/.#/3<(LO8JuM(
FE(!)-(J3%#6'/.#/3<(
First 90 pulse:
evolution
Preparation
mixing
t1
FID: C(t1,t2)
Preparation
FID: C(t1)
FT
detection
t1
FT
detection
t2
(solid rectangles
denote 90 pulses)
Spectrum I(&1,&1)
Recording a 2D data set involves repeating a pulse
sequence for increasing values of t1 and recording a
FID as a function of t2 for each value of t1.
Spectrum I(&1)
y
' 6D%
x
(For AX system:
)
Second 90 pulse:
2D contour plot
Only last (3) and (4) terms lead to observable signals. Term 3
yields diagonal peaks ('1, '1). Term 4 contains “coherence
transfer”: a ('1, '2) cross-peak modulated by '1 in t1 and by '1
in t2.
L#M(N1&+=&+(O=%+(
VP(
L#M(N1&+=&+(O=%+(
VV(
!8QJuaQÖJu(
K+I/'7&,/+(I'/7(O8Ju(
after t1:
•  )/.6('1*/'/".(:&<(/I(&..1*+1+*(3'/6/+.H(3'%.%+#%(/I(#'/..@3%&e.(
"+&7A1*"/".$<('%9%&$(N@#/"3$1+*(#/++%#,916<(
•  2&Ü%'+.(/I(#/++%#,916<(1.(#=&'&#6%'1.,#(/I(6=%(7/$%#"$%H(/+#%(&(.1+*$%(
3%&e(L%X*X;(7%6=<$M(1.(&..1*+%4;(6=%('%.6(I/$$/:(6=%(O8Ju(#/++%#,916<X(
•  N@#/"3$1+*(#/+.6&+6.(&$./(7%&."'%4X(
L#M(N1&+=&+(O=%+(
2nd 90 pulse:
1)
2)
First term is “frequency labeled” and undergo NOE/chemical exchange; second term is
eliminate by coherence selection (via phase cycling). The 3rd pulse detects the first term.
VZ(
L#M(N1&+=&+(O=%+(
VW(
G%6%'/+"#$%&'(J1+*$%(}"&+6"7(O/''%$&,/+(LGJ}OM(
O/=%'%+#%(J%$%#,/+(
•  2=&.%(#<#$1+*(&+4(*'&41%+6(3"$.%.(6/(.%$%#6a%$171+&6%(#%'6&1+(
6<3%.(/I(#/=%'%+#%(%B#16&,/+(I/'(4%6%#,/+(
after t1:
2nd 90 pulse:
1)
2)
NOESY/EXSY
•
•
•
How do one “select” I1z for detection?
A simplified scheme (the actual phase cycling is more complicated):
Scan 1: apply 2nd 90 pulse along x
Scan 2: apply 2nd 90 pulse along –x
•
•
•
At the end, subtract FIDs from two scans. Can you imagine what happens to
the above two terms?
L#M(N1&+=&+(O=%+(
Filled rectangles represent 90° pulses and open
rectangles represent 180° pulses. The delay ( =1/(2J12);
all pulses have phase x unless otherwise indicated.
VY(
)/.6(I'%`"%+6$<('%#/'4%4(1+(3'/6%1+(!)-(
Ñ,$1n%(/+%@A/+4(Oa!@G(#/"3$1+*(
QB#16&,/+(&+4(4%6%#,/+(1+(3'/6/+(#=&++%$(I/'(A%.6(
.%+.1,916<f(L:=<áM(
8+%(#'/..@3%&e(L*'/"3M(I/'(%&#=(Oa!@G(3&1'(
t%<(1+I/'7&,/+(/A6&1+%4H(#=%71#&$(.=1>.f(
}"1#e(&+4(.173$%H('&314(!)-(I%&.1A1$16<(41&*+/.1.(
&+4(.6&A1$16<(7/+16/'1+*(/I(3'/6%1+(.&73$%.(((
L#M(N1&+=&+(O=%+(
V\(
)"$,@E17%+.1/+&$(!)-(
L#M(N1&+=&+(O=%+(
PE(J3%#6'&(
V[(
~1."&$1n1+*(PE(J3%#6'&(".1+*(J6'13.(
L#M(N1&+=&+(O=%+(
ZT(
… Schematic representations of dipeptides
showing nuclei in residues i and i¡1, where i is
the residue number, that are correlated
(circled) in the HNCACB (top left) and (HB)
CBCA(CO)NNH experiments (top right). The
13CB nuclei observed in the HNCACB are
colored red, denoting the opposite phase of
signals arising from these spins relative to
phases of 13CA signals. Strip plots from the
HNCACB experiment are at the 1HN(i) and
15N(i) chemical shifts for residues His 480 to
Glu 488 of the Nedd4 WW domain (bottom).
The negative 13CB signals are represented as
red contours. Correlations between sequential
13CA/13CB resonances are indicated by
dotted lines. The asterisks (¤) in the His 480
strip identify peaks with increased intensity on
another plane. This spectrum was recorded at
500 MHz (1H frequency) on a 1-mM 15N=13Clabeled Nedd4 WW domain bound to the
unlabeled ENaCBP2 peptide in 10 mM sodium
phosphate, 90% H2O, 10% D2O, pH 6.5 at
30±C …
Multidimensional NMR Methods for Protein
Structure Determination, Kay et al., IUBMB Life
(2001).
L#M(N1&+=&+(O=%+(
http://www.protein-nmr.org.uk/assignment_theory.html
ZD(
L#M(N1&+=&+(O=%+(
ZF(
?'13$%(-%./+&+#%(2'/6%1+(!)-(
)"$,417%+.1/+&$(2'/6%1+(!)-(
Labeling of a protein with both 15N
& 13C causes almost all of the
atoms in the protein to become
observable in NMR spectroscopy.
More importantly, all of the atoms
also become scalar coupled to
each other. These homonuclear
and heteronuclear scalar
couplings are relatively large
compared to the linewidth of the
resonance lines.
Two key references:
1.  Markley (1994) Methods in Enzymology Vol 239.
2.  Bax & Grzesiek (1993), Accounts of Chemical Research, 26, 132.
L#M(N1&+=&+(O=%+(
http://www.intermnet.ua.es/inteRMNet/cursoRule/doublelabel.html
ZP(
L#M(N1&+=&+(O=%+(
http://www.intermnet.ua.es/inteRMNet/cursoRule/doublelabel.html
)"$,417%+.1/+&$(2'/6%1+(!)-(
01/7/$%#"$&'(!)-(533$1#&,/+.(
DX(G1*=@'%./$",/+(.6'"#6"'%(4%6%'71+&,/+(
FX(E<+&71#.H('%$&B&,/+(&+&$<.1.(
PX(?'&+.1%+6(1+6%'&#,/+.H(%B#16&,/+(6'&+.I%';(.31+@$&A%$1+*(
VX(J/$14(.6&6%H(7%7A'&+%(3'/6%1+.(
ZX(K+@#%$$(!)-(
WX(K7&*1+*H()-K(
((((UX(
L#M(N1&+=&+(O=%+(
http://www.intermnet.ua.es/inteRMNet/cursoRule/doublelabel.html
ZZ(
ZV(
Source: PDB
First X-ray protein structures
Hemoglobin and myoglobin, by Max Perutz
and Sir John Kendrew, respectively, in 1958
First NMR protein structure:
Proteinase inhibitor IIA by Wüthrich
lab in 1984 (published on JMB 1985).
L#M(N1&+=&+(O=%+(
ZY(
DX(G1*=@'%./$",/+(!)-(.6'"#6"'%(4%6%'71+&,/+(
L#M(N1&+=&+(O=%+(
Z\(
0&.1#(J6%3.(/I(!)-(J6'"#6"'%(E%6%'71+&,/+(
•  J&73$%(3'%3&'&,/+(&+4(4&6&(#/$$%#,/+(
•  O=%71#&$(.=1>(&..1*+7%+6.H(A&#eA/+%(&+4(.14%#=&1+(
•  !)-(1.(/+%(/I(6=%(/+$<(6:/(7%6=/4.(I/'(=1*=@'%./$",/+(
.6'"#6"'%(4%6%'71+&,/+(A%.14%.(Ö@'&<(#'<.6&$$/*'&3=<(
•  549&+6&*%.(/I(!)-(
–
–
–
–
(see a historical review by Wüthrich, Nature
Struct Biol 8, 923 - 925 (2001))
–  O=%71#&$(J=1>(K+4%B1+*(&+4(N@#/"3$1+*(#/+.6&+6.H(F+4(.6'"#6"'%.(
•  E1.6&+#%(&+4(/6=%'(.6'"#6"'&$('%.6'&1+6.H(!8QJu(
•  J6'"#6"'&$(#&$#"$&,/+.H('%.6'&1+%4(7/$%#"$&'(4<+&71#.(
•  -%c+%7%+6(&+4(9&$14&,/+(
K+(./$",/+H(7/'%(3=<.1/$/*1#&$$<('%$%9&+6(#/+41,/+.(
2'/914%(1+I/'7&,/+(/+(3'/6%1+(4<+&71#.(
-//7(6%73%'&6"'%(/'(L&$7/.6M(&+<(6%73%'&6"'%.(/I(1+6%'%.6(
E1'%#6(7/+16/'1+*(/I(A1/3=<.1#&$(&+4(A1/#=%71#&$(3'/#%..%.(
•  ]1716&,/+.(/I(!)-(
–  ?17%(#/+."71+*(&+4($&A/'(1+6%+.19%H(41h#"$6(I/'(=1*=@6='/"*=3"6;(
3'/+%(6/(="7&+(%''/'.;($%..(3'%#1.%(
–  !%%4(6/(A%(=1*=$<(./$"A$%H(.6&A$%(:16=(+%&'(7)(#/+#%+6'&,/+(
•  J/7%,7%.(x.6'&+*%y(!)-(A"o%'.(=&9%(6/(A%(".%4(
–  ]1716%4(6/(3'/6%1+(/I(7/4%'&6%(.1n%.H(q(FTT('%.14"%.(1+(*%+%'&$ (
•  Ö@'&<(&+4(!)-(#&+(A%(#/73$%7%+6&'<(
L#M(N1&+=&+(O=%+(
Also see: http://en.wikipedia.org/wiki/Protein_nuclear_magnetic_resonance_spectroscopy
Z[(
L#M(N1&+=&+(O=%+(
WT(
J7&$$(2%3,4%.(LqPT('%.14"%.M(
2'/6%1+(!)-(J&73$%(-%`"1'%7%+6.(
•  FE(2'/6/+(!)-(/+$<H(1./6/3%(%+'1#=7%+6(+/6(+%#%..&'<f(
•  0&#eA/+%(&+4(.14%#=&1+.(&..1*+%4(".1+*(O8Ju(&+4(?8OJu(
•  2'/6/+@3'/6/+(41.6&+#%(I'/7(!8QJu(
•  Qh#1%+6(3'%3&'&,/+(/I(."h#1%+6(=1*=$<(
3"'1c%4(7&6%'1&$(:16=(&33'/3'1&6%(
1./6/3%($&A%$1+*(1.(1+#'%&.1+*$<(6=%('&6%(
$171,+*(.6%3(1+(!)-(.6"41%.(/I(
A1/7&#'/7/$%#"$%.(
p22 in SDS micelles
KKKKP ARVGL GITTV LTMTT QS
–  6<31#&$$<(%B3'%..1+*(3'/6%1+.(1+(71+17&$(
7%41"7(:16=(.1+*$%(ODP(&+4(!DZ(
'%./"'#%.(L*$"#/.%(&+4(&77/+1"7(.&$6M(
•  5$$(6<31#&$(!)-(.&73$%('%`"1'%7%+6.(
&33$<H(EF8;(+/(3&'&7&*+%,#(1/+.;(#$%&+(
6"A%;(4%*&..1+*;('%I%'%+#%;(%6#(
•  0"o%'H(3G;(1/+.;(&+4(#/@./$9%+6H(6'1#e<;(
/3,71n%4(L./$"A1$16<;(.6&A1$16<;(.6'"#6"'&$(
3'/3%',%.;(%6#M(
•  }"1#e(91%:(/I(!)-(."16&A1$16<H(GJ}O(LGa
O(/'(Ga!M(
L#M(N1&+=&+(O=%+(
<NL(H'
WD(
O'%&6%.(#/''%$&,/+.(A%6:%%+(&$$(3'/6/+.(:16=1+(&(*19%+(.31+(
.<.6%7;(+/6(r".6(A%6:%%+(#$/.%(+%1*=A/'.($1e%(1+(O8JuX(
2&',#"$&'$<(".%I"$(1+(14%+,c#&,/+(/I(&71+/(&#14.f(
Finger print region (HN-Ha/Side chain protons)
of 1H-1H TOCSY
Finger print region (HN-HN) of 2D 1H-1H NOESY spectrum
L#M(N1&+=&+(O=%+(
Herrera et al PROTEINS (in press) WF(
J31+(]/#e(
Spin-echo sequence
Spin-lock sequence
COSY peaks
TOCSY peaks
“spin lock period”
Spin-lock sequence – is the Spin-echo sequence, applied continuously.
The simplest spin-lock sequence is just a continuous pulse.
L#M(N1&+=&+(O=%+(
WP(
The Spin-lock sequence makes all spins strongly coupled
(differences in chemical shifts are less than coupling constants)
E1.6&+#%(-%.6'&1+6.(I'/7(!8Q(
]&'*%'(2'/6%1+.(
•  E&6&(5#`"1.1,/+H(
•  OJ(&..1*+7%+6('%`"1'%.(4/"A$<@$&A%$%4(3'/6%1+.(
•  5"6/7&,#(&..1*+7%+6(/>%+(I%&.1A$%H(#/''%#6(&..1*+7%+6(&(
#'1,#&$(.6&',+*(3/1+6f(
High resolution 1H-15N HSQC
High resolution 1H-13C HSQC (aliphatic)
High resolution 1H-13C HSQC (aromatic)
Backbone: HNCA
HN(CO)CA
HNCO
HCACO
HN(CA)CO
CBCA(CO)NH
HNCACB
HBHA(CBCACO)NH
HN(CA)HA
3D
NOESY
3D 13C NOESY
15
4D N, 13C NOESY
4D 13C, 13C NOESY
3D 15N, 15N NOESY
3D 15N HSQC-NOESY-HSQC
(take two NOESY spectra at 2 or
more mixing times when possible)
Side chain: 15N TOCSY-HSQC
C(CO)NH-TOCSY
H(CCO)NH-TOCSY
CCH-TOCSY
CCH-COSY
HCCH-TOCSY
HCCH-COSY
Aromatic
1H spectra (2QF COSY and 2Q) for resonances
within aromatic rings
1H NOESY to connect Hd with Hb (marked high
intensity NOEs)
(Hb)Cb(CgCd)Hd
(Hb)Cb(CgCdCe)He
(HC)C(C)CH-TOCSY
Methionine
HMBC to assign methyl group
•
•
•
•
!8Q(A"1$4"3(d(Da'W(
5#6"&$(.$/3%(&$./(4%3%+4.(/+(
4<+&71#.;(.31+(41o".1/+;(.6'/+*(
#/"3$1+*(%6#(
!8QJu(1.(+/1.<f(
Ñ,$1n%(e+/:+(41.6&+#%.H(1+6'&@
'%.14"%(/+%.;(F+4(.6'"#6"'%.(
•  !8Q(&..1*+7%+6.H(/9%'$&3.($%&4(6/(&7A1*"/".(!8Q.à(16%'&,9%(
&..1*+7%+6(&..1.6%4(:16=(.6'"#6"'&$(I%%4A&#e.f((((
•  01++1+*(!8Q(1+6%+.1,%.H($&'*%("+#%'6&1+,%.(&+4(/+$<(`"&$16&,9%f(
;?1""
Incomplete excerption from Wright/Dyson lab manual (The Scripps Research Institute).
L#M(N1&+=&+(O=%+(
•  O&$1A'&,/+(/I(!8Q(
15N
(
(/&"$/1)*$'
'9&";/)%B#* (wI/'(3'/6%1+(:a()'qFT(eE&(
.6'/+* (
7%41"7(
:%&e (
(DX\@FXY({
(DX\@PXP({
(DX\@ZXT({
(.6'/+*(1+6%+.16<(1+(.=/'6(67(LdZT(7.wM(!8QJu(
(:%&e(1+6%+.16<(1+(.=/'6(67(LdZT(7.wM(!8QJu(
(/+$<(91.1A$%(1+($/+*%'(71B1+*(,7%(!8QJu(
WZ(
L#M(N1&+=&+(O=%+(
(
WW(
3D 1H-15N NOESY-HSQC spectrum of a dimeric GpA mutant
PE(!8QJu@GJ}O(
•  FE(DG@DG(!8QJu(#&+(A%(9%'<((
#'/:4%4X(
•  5441,/+&$(417%+.1/+(=%$3.(6/(
41.3%'.%(6=%(3%&e.(&+4(I&#1$16&6%(
&..1*+7%+6(
•  !8QJu(I/$$/:%4(A<(GJ}O(
L#M(N1&+=&+(O=%+(
WY(
http://www-bioc.rice.edu/~mev/spectra3.html
L#M(N1&+=&+(O=%+(
W\(
J6'"#6"'&$(O&$#"$&,/+(
5441,/+&$(J6'"#6"'&$(-%.6'&1+6.(
•  5$$(.6'"#6"'&$('%.6'&1+6.(173$%7%+6%4(A<(3%+&$6<(I"+#,/+.(
•  O=%71#&$(.=1>(&+&$<.1.H(OJK(
•  G<4'/*%+(A/+4.H(EF8(%B#=&+*%(%B3%'17%+6.(6/(14%+,I<(.$/:(
%B#=&+*1+*(&714%.(L6=/.%(1+9/$9%4(1+(=@A/+4.M(
•  N@#/"3$1+*(#/+.6&+6.(
Experiment
Coupling constant
3J
HNHA
HN,Ha
3J
HA(CA)HB COSY
Ha,Hb
3J
HNHB
HN,Hb
2QF COSY
JHN,Ha, 3JHa,Hb
13C-{13CO} spin-echo difference ct-HSQC
3J
Cg,CO
13C-{15N}
spin-echo difference ct-HSQC
3J
Cg,N
L#M(N1&+=&+(O=%+(
V = "VMM + "VNMR
!
Information
(%
c1, bH stereos
c1, bH stereos
confirmation
Ile, Thr )1, Val )1,
C* stereos
Ile, Thr )1, Val )1,
C* stereos
W[(
Distance rij
•  J6'"#6"'&$(#&$#"$&,/+('%@#&.6%4(&.(*$/A&$(%+%'*<(71+171n&,/+a
#/+I/'7&,/+&$(.%&'#=(3'/A$%7(L:=1#=(1.(7/$%#"$&'(4<+&71#.(&$$(&A/"6fM(
•  -%.6'&1+%4(7/$%#"$&'(4<+&71#.(:16=(.17"$&6%4(&++%&$1+*(6/(*%+%'&6%(.%6.(
/I(.6'"#6"'%.(6=&6(7&B17&$$<(.&,.I<(&$$('%.6'&1+6.(
•  ?:/(e%<(3/3"$&'(!)-(.6'"#6"'&$(#&$#"$&,/+(./>:&'%(
–  Ou5!5aEu5!5H(=Ü3Haa:::X$&.Xr3a3'/4a#<&+&a%*(
–  O!JH(=Ü3Haa#+.@/+$1+%X/'*a9DXFDa(
–  Ö3$/'@!KGH((=Ü3Haa+7'X#16X+1=X*/9aB3$/'@+1=a(
L#M(N1&+=&+(O=%+(
YT(
Internal Coordinate MD
L#M(N1&+=&+(O=%+(
YD(
L#M(N1&+=&+(O=%+(
YF(
-%c+%7%+6(1+(173$1#16(./$9%+6(#&+(A%(".%4(6/(/A6&1+((
+&,9%@$1e%(7/4%$.(I'/7($1716%4(!)-(4&6&X(
-%c+%7%+6(|(~&$14&,/+(
•  !)-('%.6'&1+6(91/$&,/+(.6&,.,#.H(
.%$I@#/+.1.6%+#<(
•  O/+9%'*%+#%(L3'%#1.1/+MH(#&+(A%(
71.$%&41+*(
•  2-8OGQOtH(2E0(.6&,.,#.(/+(
*%+%'&$((a+(41.6'1A",/+.(
•  -%c+%7%+6(".1+*(&441,/+&$(
1+I/'7&,/+(I'/7(
–  Q731'1#&$(3'/6%1+(I/'#%(c%$4H(
./$9%+6(%o%#6.(
V = "VMM + "VNMR + "VOther
!
–  5441,/+&$(%B3%'17%+6&$(4&6&H(
!)-(&+4(+/+@!)-f(
–  -%.14"&$(413/$&'(#/"3$1+*(L-EOM((
PDB: 2KI7; Xu et al, JMB (2009)
L#M(N1&+=&+(O=%+(
-%c+%7%+6(/I(
)&$6/.%@01+41+*(
2'/6%1+(L)02M(
•  370 residues, 42 kDa !
•  1943 NOE, 45 hydrogen
bonding and 555 dihedral angle
restraints.!
•  Average backbone RMSD to
X-ray structure is 5.5 Å (a).!
•  Improved to 3.3 Å with 940
additional dipolar coupling
based restraints (b).!
Ref. Mueller et al., JMB 300, 197 (2000)!
YP(
Cost: ~12h wall time using 16 Intel 2.4GHz CPUs!
K73$1#16(J/$9%+6(-%c+%7%+6(-%."$6.(
•  All NOE and dihedral angle
restraints were used.!
•  48 replicas were simulated at
300 to 800 K until converged.!
•  Total of 1.0 ns REX/GB
simulation.!
!
!
!
!
!
!Initial
RMSD to X-ray (Å) a!
Global !
!
!
!
!4.3±4.1
N-domain !
!
!
!2.5±2.1
C-domain
!
!
!
!3.0±3.2
(/+ space: residues (%)!
Most favored
!
!
!72.2 !
Additionally allowed !
!22.8 !
Generously allowed !
!3.8 !1.6!
Disallowed !
!
!
!1.2 !
Violation statistics!
RMSD of NOEs (Å) !
!0.0047
NOE violations ( > 0.2Å)
!2.85 !
RMSD of angles (in degrees) !0.53 !
a
!Final!
!2.3±2.6!
!2.2±1.4!
!2.0±1.9!
!84.3!
!13.3!
!0.8!
!0.014!
!4.42!
!6.25!
Backbone RMSD with respect to PDB:1dmb
shown. Global: residues 6-235 and 241-370;
N-domain: 6-109 and 264:309; C-domain:
114-235, 241-258 and 316-370.!
-%3'%.%+6&,9%(J6'"#6"'%.H()02(
5.8 Å
!
!
!
!
!
! 2.9 Å!
5.7 Å
!
!
!
!
!
!
5"6/7&,#aG1*=(?='/"*=3"6(!)-(J6'"#6"'%(E%6%'71+&,/+(
“The automation of protein structure determination using NMR is coming of age. The tedious processes of
resonance assignment, followed by assignment of NOE (nuclear Overhauser enhancement) interactions
(now intertwined with structure calculation), assembly of input files for structure calculation, intermediate
analyses of incorrect assignments and bad input data, and finally structure validation are all being
automated with sophisticated software tools. The robustness of the different approaches continues to deal
with problems of completeness and uniqueness; nevertheless, the future is very bright for automation of
NMR structure generation to approach the levels found in X-ray crystallography. Currently, near
completely automated structure determination is possible for small proteins, and the prospect for mediumsized and large proteins is good.”
3.5 Å!
“Automation of NMR structure determination of proteins”, Altieri and Byrd, Curr Opin Struct Biol (2004)
RMSD values: from X-ray (PDB:1dm); backbone atoms of residues 6-235 and 241-370!
O5!EKE(2'/6/#/$(
Y\(
cycle 1
FX(2'/6%1+(E<+&71#.(I'/7(-%$&B&,/+(5+&$<.1.(
Relaxation parameters (T1, T2,
NOE) determined mainly by
molecular tumbling and also
depends on internal dynamics
They thus report on internal
dynamics, even though not
always in obvious ways!
Final cycle
Wuthrich and coworkers, JMB (2002)
L#M(N1&+=&+(O=%+(
L#M(N1&+=&+(O=%+(
Y[(
blue: with Cu2+; red: without
~1$$&+"%9&(%6(&$X;(xK+#'%&.%(1+(6=%(#/+I/'7&,/+&$(â%B1A1$16<(
/I(#F@71#'/*$/A"$1+("3/+(#/33%'(A1+41+*H(5(3/..1A$%('/$%(
L#M(N1&+=&+(O=%+(
\T(
I/'(#/33%'(1+(41&$<.1.@'%$&6%4(&7<$/14/.1.y;(2'/6X(J#1(LFTT[MX(
J3%#6'&$(E%+.16<(&+4(K+6%'+&$()/,/+.(
}"&+,6&,9%(5+&$<.1.(/I(!)-(-%$&B&,/+(
•  ?=%(.3%#6'&$(4%+.16<(I"+#,/+(1.(6=%(C/"'1%'(6'&+.I/'7(/I(6=%(&+*"$&'(&"6/@
#/''%$&,/+(I"+#,/+;(OL6M;(/I(6=%(!@G(A/+4(9%#6/';(
•  Å16=(L3'/6/+@3'/6/+M(#'/..@'%$&B&,/+(."33'%..%4;(&714%(!DZ(
'%$&B%.(3'17&'1$<(4"%(6/(413/$&'(1+6%'&#,/+(:16=(6=%(41'%#6$<(
&Ü&#=%4(DG(.31+(&+4(6='/"*=(DZ!(O=%71#&$(J=1>(5+1./6'/3<X(
•  -%$&B&,/+(3&'&7%6%'.(4%6%'71+%4(A<(.3%#6'&$(4%+.1,%.H((
•  ?=%(&"6/@#/''%$&,/+(I"+#,/+(4%3%+4.(/+(A/6=(/9%'&$$(6"7A$1+*(&+4(
1+6%'+&$(4<+&71#.X(5.."71+*(OL6M(R(O/L6M(OKL6M;(1I(6"7A$1+*(7"#=(.$/:%'X(
•  K+(./@#&$$%4(x7/4%$@I'%%y(&+&$<.1.(L]13&'1(&+4(Jn&A/;(D[\FM;(1+6%'+&$(
4<+&71#.(#=&'&#6%'1n%4(A<(L7/,/+M(7/4%$@I'%%(3&'&7%6%'.;(1+#$"41+*((
–  *%+%'&$1n%4(/'4%'(3&'&7%6%'.(L(MH(&73$16"4%.(/I(6=%(1+6%'+&$(7/,/+.(
–  Qo%#,9%(,7%(#/+.6&+6.(L"MH(,7%@.#&$%.(/I(6=%(1+6%'+&$(7/,/+.(
Sample CI(t) with S2=0.9, "e=50 ps
rNH is the length of the N-H bond; () is the CSA of 15N;
$H and $N are the Larmor frequencies of 1H and 15N
Chen et al., JBNMR (2004) and references therein.
L#M(N1&+=&+(O=%+(
\D(
)/4%$@C'%%(5+&$<.1.(
Time (ps)
L#M(N1&+=&+(O=%+(
\F(
Assumption of uncoupled tumbling and internal motions in model-free
analysis likely leads to an underestimation of motions on ns timescales.
•  )%&."'%(?D;(?F(|(!8Q;(6<31#&$$<(&6(6:/(c%$4.(L%X*X;(ZTT()Gn(&+4(WTT()GnM(
•  C/'(%&#=(!DZ;(c6(L6='%%(/'(.1BM('%$&B&,/+(4&6&(3/1+6.(6/(6/(/A6&1+(
*%+%'&$1n%4(/'4%'(3&'&7%6%'.(LJM(&+4(%o%#,9%(,7%(#/+.6&+6.(L"MX(
•  ?=%.%(4<+&71#.(3&'&7%6%'.(`"&+,I<(3'/6%1+(1+6%'+&$(4<+&71#.(&+4(#&+(A%(
".%(6/("+4%'.6&+4(6=%('/$%(/I(1+6%'+&$(4<+&71#.(1+(I/$41+*(&+4(A1+41+*X(
NMR PROBES OF MOLECULAR DYNAMICS: Overview and Comparison with
Other Techniques, Palmer,
Annu Rev Biophys Biomol Struct (2001).
L#M(N1&+=&+(O=%+(
\P(
A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling
15N NMR relaxation, Tugarinov et al, JMB (2002).
L#M(N1&+=&+(O=%+(
\V(
2'/6%1+(4<+&71#.(1+(%+n<7%(#&6&$<.1.(
mesoAdk
Schnell et al, Annu Rev
Biophys Biomol Struct
(2004)
thermoAdk
The synergy between structure and dynamics is
essential to the function of biological
macromolecules. …. Here we show that pico- to
nano-second timescale atomic fluctuations in
hinge regions of adenylate kinase facilitate the
large-scale, slower lid motions that produce a
catalytically competent state. The fast, local
mobilities differ between a mesophilic and
hyperthermophilic adenylate kinase, but are
strikingly similar at temperatures at which
enzymatic activity and free energy of folding are
matched.
thermoAdk @ 20C
50C
80C
The connection between
different timescales and the
corresponding amplitudes of
motions in adenylate kinase
and their linkage to catalytic
function is likely to be a
general characteristic of
protein energy landscapes.
A hierarchy of timescales in protein dynamics is linked
to enzyme catalysis, Kern and coworkers, Nature (2007)
L#M(N1&+=&+(O=%+(
\Z(
L#M(N1&+=&+(O=%+(
O/+I/'7&,/+&$(Q+6'/3<(1+(2'/6%1+(K+6%'&#,/+(
O/"3$%4(01+41+*(&+4(C/$41+*(
Here we employ changes in conformational dynamics as a
proxy for corresponding changes in conformational entropy.
We find that the change in internal dynamics of the protein
calmodulin varies significantly on binding a variety of target
domains. Surprisingly, the apparent change in the
corresponding conformational entropy is linearly related to
the change in the overall binding entropy. This indicates that
changes in protein conformational entropy can contribute
significantly to the free energy of protein–ligand association.
\W(
intra-well
rotamer conv.
“Encounter Complex”
Wand and coworkers, Nature (2001); Nature (2007)
(sidechain methyl axial)
pKID/KIX
L#M(N1&+=&+(O=%+(
\Y(
Wright and coworkers, Nature (2007); COSB (2009)
L#M(N1&+=&+(O=%+(
\\(
~1."&$1n1+*(6'&+.1%+6(%9%+6.(1+((
&71+/@6%'71+&$(&"6/3'/#%..1+*(/I(GK~@D(3'/6%&.%(
PX(J31+@$&A%$1+*(&+4(?'&+.1%+6(K+6%'&#,/+.(
•  2&'&7&*+%,#(#%+6%'.(%+=&+#%(!)-('%$&B&,/+;(6<31#&$$<("+4%.1'%4X(
•  2&'&7&*+%,#('%$&B&,/+(%+=&+#%7%+6(L2-QM(#/"3$%4(:16=(.16%@41'%#6%4(
.31+@$&A%$1+*(LJEJ]M;(=/:%9%';(3'/914%.(41.6&+#%(1+I/'7&,/+("3(6/(PZ({X(
–  5$$/:.(/+%(6/(4%6%#6($/+*@'&+*%(/'4%'1+*(&+4(6'&+.1%+6(.6'"#6"'%.f((((
Structural Reorganization of *-Synuclein
Structures of typical nitroxide-based spin probes
Fanucci & Cafiso, COSB (2006); Wu et al, JMB (2009)
L#M(N1&+=&+(O=%+(
\[(
ZX(K+@O%$$(!)-(
PREs were measured on a 1:1 mixture of 0.2+mM U-[2H/13C/15N]-labelled SFNFPR(D25N) and spinlabelled SFNFPR(D25N) at natural isotopic abundance.
Tang et al, Nature (2007)
L#M(N1&+=&+(O=%+(
[T(
A putative heavy-metal binding protein TTHA1718 from Thermus
thermophilus HB8 overexpressed in Escherichia coli cells.
a, A superposition of the 20 final structures of TTHA1718 in living E. coli cells, showing the
backbone (N, Calpha, C') atoms. b, A superposition of the 20 final structures of purified
TTHA1718 in vitro. c, A comparison of TTHA1718 structures in living E. coli cells and in vitro.
The best fit superposition of backbone (N, Calpha, C') atoms of the two conformational
ensembles are shown with the same colour code in a and b. d, Secondary structure of
TTHA1718 in living E. coli cells. The side chains of Ala, Leu and Val residues, the methyl
groups of which were labelled with 1H/13C, are shown in red. e, Distance restraints derived
from methyl-group-correlated and other NOEs are represented in the ribbon model with red
and blue lines, respectively.
a, Scheme of the in-cell NMR
experiments using E. coli cells. b, The
1H–15N HSQC spectrum of a
TTHA1718 in-cell NMR sample
immediately after sample preparation.
c, The 1H–15N HSQC spectrum after 6
h in an NMR tube at 37 °C. d, The 1H–
15N HSQC spectrum of the supernatant
of the in-cell sample used in b and c.
Protein structure determination in living cells by in-cell NMR spectroscopy, Sakakibara et al, Nature (2009).
L#M(N1&+=&+(O=%+(
[D(
Protein structure determination in living cells by in-cell NMR spectroscopy, Sakakibara et al, Nature (2009).
L#M(N1&+=&+(O=%+(
[F(
WX()-K(
x7&*+%,#('%./+&+#%(6/7/*'&3=<y(
C1'.6()-(17&*%(1+(D[YP(
FTTP(!/A%$(2'1n%(
E%6%#6(?D(/'(?F('%$&B&,/+(/I(:&6%'(
3'/6/+.(1+(A/4<(
•  O/+6'&.6(&*%+6(6/(%+=&+#%(6=%(#/+6'&.6(
L1+#'%&.%(6=%(.3'%&4(/I(?Da?F(1+(,.."%.M(
•  0T(d(DXZ(?(LWT()GnM(
•  QB6%+.19%(".%(/I(7&*+%,#(c%$4(*'&41%+6(
I/'(3$&+%(.%$%#,/+(
•
•
•
•
Additional reading: http://www.cis.rit.edu/htbooks/mri/
L#M(N1&+=&+(O=%+(
[P(