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Lecture 13 M230
Feigon
 Nucleic acids in water
 Chemical exchange
 Chemical shift mapping
Reading resources
Evans Chap 1.3 Kinetics and chemical exchange
HSQC
heteronuclear single quantum correlation
1H-13C HSQC -- correlates proton with attached carbon
1H-15N HSQC -- correlates proton with attached nitrogen
Gotfredsen, et. al,
J. Am. Chem. Soc.,
120, 4281 (1998)
155
Folded
peaks
13C resolves:
AH2 from H8/H6
CH5
UH6
RNA Ribose
H4’;H5’,H4”;2’,3’
RNA CH5;UH5
190
1
C G C G A A T T C G C G! in H O
2
G C G C T T A A G C G C!
10°C
τm = 100 msec
aromatic, amino
NOESY with 13 31 observe pulse
90 - t1 - 90 - τm - acquire
13 31
imino
NOESY spectra of DNA in H2O
assignment of exchangeables
C G C G A A T T C G C G!
Presat NOESY in H2O
B1) imino-AH2 NOEs
Get strong NOE between imino and AH2
(~2.2Å). Can identify A’s by this strong
sharp NOE. If iminos assigned, have
assigned AH2.
B2) imino-amino NOEs
aminos ~5.5 - 9 ppm
H-bonded amino in pair (2) usually ~2ppm
lower field than non-H-bonded amino (1)
C aminos: slow exchange
2 sharp resonances
A aminos: intermediate exchange
usually can see both (2)
and (1), but weaker than C’s
G aminos: intermediate-fast exchange
usually a broad, coalesced
peak, hard to see A) imino-imino NOEs
Can assign iminos sequentially along
strand via 1D or 2D NOEs
2
“Formation of a stable triplex from a single DNA strand” Sklenar & Feigon, Nature (1990)
Crosspeaks between iminos give sequential
connectivities along each ‘strand’.
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The base pairing scheme can be determined from specific NOEs
observed between imino and amino, H2, and H8 resonances.
Today we would also 13C,15N-label the DNA or RNA, and
detect the Watson-Crick and Hoogsteen paired iminos
directly using HNN-COSY experiments
Portion of spectrum showing crosspeaks between
acceptor N (N1 or N7) and H from donor UN3H;
Donor N (UN3) are ~155-165 ppm
Kim, et al JMB 2008 Wild-type human telomerase pseudoknot
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Conformational equilibria; chemical exchange
We have seen that intramolecular triplex and duplex + ss conformations in equilibrium
give rise to two separate sets of peaks in NMR spectra. This is because the molecules
are in slow exchange on the NMR time scale. What does this mean?
Example:
duplex + ss
kdt
ktd
Example:
triplex
kHC
kCH
C
fast
intermediate
slow
H
T
pH
d+ss
t
δ
δ
ktd, kdt << πΔνdt
Two separate sets of peaks;
Relative intensities depends on
fraction in each form
Hz (sec-1)
H
δ
C
more complicated;
kHC, kCH >> πΔνHC
shifts and broadens
One peak; Chemical shift depends on
fraction in each form.
δob = δHa + (1-α)δCa
Fast or slow exchange on NMR time scale depends on rate relative to chemical shift difference (Δν)
between the two resonances.
Hz, sec-1
This will vary with
field (500 MHz → “slower” than 200 MHz)
which resonances (Δνxy ≠ ΔνAB)
temperature (may change kinetics)
Chemical exchange effects on the NMR spectrum
Slow exchange.
k << Δω
τ >> 1/Δω
τ is avg lifetime in
the state
Intermediate exchange.
k ~ Δω.
τ ~ 1 /Δω
Fast exchange.
k >>> Δω
τ << 1/Δω
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Helix-coil transition
Can monitor the transition H kHC C
kCH
by NMR.
Recall: Chemical shifts in nucleic acids are dependent on ring current shifts.
As bases become unstacked, will generally shift downfield.
For ds DNA or RNA helix-to-coil transition is intermediate
to fast exchange. If fast, can plot chemical shift vs T to get
melting temp. (Tm) and other thermodynamic parameters.
δ
Example: DNA hairpin
T ºC
ATCCTA!T!T!
......!
TAGGAT!T!T!
6.0
ATCCTATTTTTAGGAT!
1.8
Compare curves for
TCH3 in stem and loop
δ(ppm)
5.8
1.6
5.6
1.4
280
300
320
340 0 K
280
300
320
340 0 K
Chemical shift mapping: identification of interacting surface
slow exchange
1H-15N
15N-protein+
HSQC
ligand 1:0.5
1H
5
free 15N-protein
1
6
1H
1
2
6
1H
1
15N
2
4
2
3
15N
titration
1
4
+ ligand
5
free
6
1H
1
5
2
3
15N
fast exchange
15N-protein
6
1
3
complex
5
4
3
4
3
15N-protein-ligand
6
5
4
2
15N
4
2
3
6
5
bound
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Chemical shift mapping
Nucleolin RRM12 free and bound to RNA
Free
Bound
15N
15N
1H
1H
Overlay of free and bound
15N
1H
Chemical shift changes of nucleolin RRM12 upon binding RNA
650
600
550
500
450
|ΔN|+|ΔH|
Hz
400
350
300
250
200
150
100
50
0
10
20
β1
30
40
α1
50
β2
60
70
β3
α2
RRM1
80
90 100 110 120 130 140 150 160 170
β4
RRM1
Linker
β1
α1
β2
β3
α2
RRM2
Linker
Helix 1 Helix 1
RRM2
Linker
|ΔN|+ |ΔH|
β2-β3 loop
β2-β3 loop
180°
β4
> 300 Hz
200 – 300 Hz
120 – 200 Hz
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EXAM: Tuesday 3-5
•  Designed to be one hour, you will have two hours.
•  All homework problems are old exam problems.
•  We will provide chemical shift tables.
You can bring:
•  ONE page of notes. Must be written by hand.
•  A ruler or straight edge.
•  A simple calculator.
GOOD LUCK!!!!
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