Conformational Dynamics of
Telomeric DNA via Single
Molecule FRET
D. S. Kim
School of Physics, Seoul Nat’l Univ., South Korea
Nov. 2nd 2005
Contents
1. Introduction
Single Molecule Spectroscopy & FRET
Telomere & G-quadruplex
2. Conformation Dynamics
Human Telomeric DNA
Mutated Human Telomeric DNA
Protein Binding
4-quartet G-quadruplexes
3. Summaries
Why Single Molecule?
• Remove the ensemble average effect
► Construction of a frequency histogram of the actual
distribution of values for an experimental parameter
► Give more information than standard ensemble
measurements
• Observation of exact dynamics between conformational
states without synchronization
Single Molecule Spectroscopy
AFM
PNAS, 96, 11288, 1999
Electronics
Biop. J., 87, 3205 2004
Optical Tweezers
Nature, 365, 721, 1993
Fluorescence
Science, 283, 1676, 1999
FRET
Fluorescence Resonance Energy Transfer
Energy Transfer by dipole-dipole interaction
between two fluorophores, donor and acceptor
Energy Transfer Efficiency
Ia
1
~
E=
6
Ia + Id
1 + (R / R0 )
Energy Transfer Range
Single Molecule FRET (smFRET)
Excitation light
Acceptor fluorophore
Donor fluorophore
Biological molecule
Schematic diagram of smFRET
Fluorescence intensity change by
conformational changes of a single molecule
S. Weiss, Science, 283, 1676, 1999
Experimental Setup for smFRET
Prism-type TIR
532 nm laser
Mirror
Excitation laser
Lens
Quartz slide
Mirror Dichroic
sample
Lens
Microscope
Evanescent field
Fluorescence
Lens
Objective
CCD
Long-pass filter
Prism
Index matching oil
Telomere
&
G-quadruplex
Telomere
Definition
The end regions of the chromosomes
- non-coding DNA: tandem repeat of short sequences
- associated proteins
Function of telomere
Protection of the chromosome ends from degradation
- end-to-end fusion
- damage by exo-nucleases
Shortened as the cell division goes on
- Apoptotic process
Human Telomere Structure
t-loop
Tandem repeat of GGGTTA
D-loop
S. Neidle & G. Parkinson, Nature Drug Discovery, 1, 383, 2002
Guanine Tetrad (G-quartet)
A square co-planar bonding of 4 guanine bases,
where each base is both the donor and acceptor of
2 hydrogen bonds with its neighbors
- Hoogsteen bonding
Guanine tetrad (G-quartet)
T. Simonsson, Biol. Chem., 382, 621, 2001
Human G-quadruplex
• Human telomeric DNA: short tandem repeat (GGGTTA)
• Polymorphisms of Human G-quadruplex
Anti-parallel
Na+ based conformation from NMR
Parallel
K+ based conformation from x-ray
crystallography
Anti
Anti
Na+
K+
Syn
Na+
Y. Wang et al. Structure, 1, 263, 1993
K+
G. N. Parkinson et al., Nature, 417,
877, 2002
13
Conformational Dynamics
Experiments
Sample
G-quadruplex part
5’-Cy5-GGGTTAGGGTTAGGGTTAGGG
AGAGGTAAAAGGATAATGGCCACGGTGCG3’-biotin
Complementary stem part
5’-CGCACCGTGGCCATTATCCTT
T*TACCTCT-3’
(T*: TMR-labeled Thymine)
Immobilization of samples
BSA-biotin & Streptavidin
Biotin-GQ
Streptavidin
BSA-biotin
Quartz
Heterogeneity of G-quadruplex
750
500
250
Long-lived
species
0.8
0.4
0.0
700
350
500
Short-lived
species
0.8
0.4
400
300
200
0.0
1000
FRET F.I
Dwell time analysis
for mixed species
# of events
FRET F.I
FRET F.I
At 2 mM K+
500
Mixed
species
0.8
0.4
0.0
0
150
300
450
600
Time (sec)
Heterogeneity !
750
900
100
0
40
80
120
Time (sec)
160
Double exponential fitting
t1 ~ 110 sec & t2 ~ 15 sec
Titration of K+ at RT
Population histogram
100 mM
700
0
K+
Before transition, the 2mM
molecule
must go through the low FRET
600
10 mM
0
450
500
# of molecules
100
2 mM
140
0.1 mM
0
3 distinct peaks !
300
200
100
0 mM
100
0
400
Low FRET
▼
Unfolded single-strand overhang
0
0
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
FRET Efficiency
0 .0
0 .2
0 .4
0 .6
0 .8
FRET Efficiency
1 .0
donor
acceptor
Time trace at 2mM K+
1000
FRET F.I.
# of molecules
150
800
600
400
200
0
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Time (sec)
120
140
160
Dynamics of G-Quadruplex
G-quadruplex
maintains one conformation
G-quadruplex fluctuates
fast
G-quadruplex
maintains one conformation again
time
or
Trend by Concentration at RT
Anti
Na+
Syn
Anti
Na+
Na+
Syn
Na+
Low
Conc. Long-lived
Unfolded
All species are mixed
at 2 mM K+
Long-lived
Anti-parallel
High
Conc.
Trend by Temperature
2 mM K+
Anti
Anti
Na+
Na+
Syn
Syn
Na+
Na+
Long-lived
Low Folded/Unfolded
Short-lived
Occurrence
Long-lived
Unfolded
Temp.
100 mM K+
Anti
Na+
Syn
Na+
Anti
Na+
Syn
Na+
Long-lived
Anti-parallel
Long-lived
Parallel
Short-lived
Occurrence
High
Temp.
Mutated Human G-quadruplex
Why Mutation?
The most important role of G-quadruplexes
► Protection of telomere
One way for protection
► Prevention from the binding of external proteins with
G-quadruplexes
Comparison between wild-type and mutated G-quadruplexes
► Confirmation of the role of G-quadruplexes
► Observation of the protein binding mechanism
Single-base Mutation
Single-base mutated G-quadruplex part
5’-Cy5-GGGTTAGGGTTAGTGTTAGGG
AGAGGTAAAAGGATAATGGCCACGGTGCG-3’-biotin
Complementary stem part
5’-CGCACCGTGGCCATTATCCTT T*TACCTCT-3’
(T*: TMR-labeled Thymine)
Guanine → Thymine
Schematic diagram of mutated G-quadruplex
Time Traces of Mutated Sample
F.I.
800
400
FRET
0.8
0.4
500
250
F.I.
FRET
F.I.
FRET
F.I.
FRET
300 mM
100 mM
0.8
0.4
600
300
30 mM
0.8
0.4
300
150
10 mM
0.8
0.4
0
20
40
60
80
Time (sec)
100
120
donor
acceptor
FRET
Telomere Binding Protein
AtWhy1 (One of Whirly protein family)
1. One of plant transcription factors for defense gene regulation
2. Single stranded binding protein
3. Binding with telomeric DNA
► The exact binding mechanism is not revealed yet.!
※ The sample is obtained from I. K. Chung in Yonsei Univ.
3D structure of Whirly protein
D. Desveaux et al.,Trends in Plant Sci., 10, 95, 2005
# of molecules
Protein Binding (200 mM K+)
600
450
300
150
New peak
appearance
Normal AtWhy1
600
AtWhy1 N-terminal
400
200
Non-binding
250
No protein
125
0
0.0
0.2
0.4
0.6
FRET Efficiency
0.8
1.0
# of molecules
Mutation vs. Wild-type
225
150
75
Before protein
500
After protein
Mutated
G-quadruplex
250
200
Before protein
100
Wild-type
600
300
0
G-quadruplex
After protein
0.0
0.2
0.4
0.6
0.8
FRET Efficiency
1.0
Schematic Diagram
Wild-type G-quadruplex
Mutated G-quadruplex
AtWhy1
AtWhy1
AtWhy1
AtWhy1
AtWhy1
1
hy
W
At
K+
K+
AtWhy1
4-Quartet G-quadruplex
4-quartet G-quadruplex
K+
K+
One more
quartet
K+
K+
K+
What is the effect of additional quartet on the conformational dynamics?
4-quartet G-quadruplex: (GGGGTTTT)3GGGG
► Biologically, this is the telomeric sequences of Oxytricha
► Oxytricha and its telomere model
C.H.Kang et al., Nature, 356,126, 1992
Time Traces at 2 mM K+
300
150
0.8
0.4
400
200
0.8
0.4
500
250
0.8
0.4
400
200
0.8
0.4
0.0
Long-live species are very dominant!
(> 99%)
No heterogeneity !
0
200
400
600
800
1000
Survival Time Analysis at 2 mM K+
• Survival Time Analysis
Measure how long the molecules survive before
transition or photo-bleaching
50
20
40
15
30
10
20
5
10
0
0
200
400
600
800
1000
Unfolded survival time
T1 ~ 363.6 sec
0
0
200
400
600
800
Folded survival time
T1 ~ 690.9 sec
This dwell time is at least 5 fold larger
than for the human telomeric DNA.
1000
Crystal Structure of Oxytricha’s Telomere
K+ formation of two d(GGGGTTTTGGGG)s
Loop
Binding
Between
quartets
Loop
Binding
S. Haider, G. N. Parkinson, & S. Neidle, JMB, 320, 189~200 (2002)
Coordination bonding of more than two K+ ions
→ higher stability than human telomeric DNA
► High stability of Oxytricha’s telomeric DNA
Summaries
Via single molecule FRET
• Human telomeric DNA.
- Confirm the coexistence of parallel and anti-parallel
conformations.
- Heterogeneity of conformational dynamics.
• Mutated human telomeric DNA.
- Short-lived folded species are dominant.
- Easy to bind with single-strand protein.
► G-quadruplex is a protective structure of telomere.
• 4-quartet G-quadruplex.
- More stable than 3-quartet G-quadruplex.
► Coordination bonding of more than 2 cations.
► High stability of Oxytricha’s telomere.