Gas-Phase DNA Helix Conformations
Transcript of Gas-Phase DNA Helix Conformations
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GasGas--Phase DNA Helix Conformations Phase DNA Helix Conformations
Erin Shammel Baker, Jennifer Gidden, Erin Shammel Baker, Jennifer Gidden, Alessandra Ferzoco, Alessandra Ferzoco,
Thomas Wyttenbach Thomas Wyttenbach and Michael Bowersand Michael Bowers
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Outline
• Experimental Method
• Theoretical Method
• Instrumentation
• DNA Background
• DNA Helix Conformations in Gas-Phase
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Drift cell
E
p(He)
IonFelFfriction
v = const.v = K EK = ion mobility
Concepts
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K = f (T, p, q, µ, σ)
T = temperaturep = pressureq = ion chargeµ = reduced massK = ion mobilityσ = collision cross section
σ = f ( )He–ion interactionIon shape
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in out
Drift cell
E
1–5 torr He
Ion mobility method
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IonSource
IonSource MS1MS1 Drift
CellDriftCell MS2MS2 DetectorDetector
Ion Mobility Instrumentation
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Ion Sources
• Matrix Assisted Laser Desorption Ionization (MALDI)
• Electrospray Ionization (ESI) to MS
to MS
LASER
E
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MALDI-TOF
Source
TOFDrift Cell
Quadrupole
TOF DetectorGlassl = 20 cmp = ~1.5 torr He
hν
Erin S. Baker, Jennifer Gidden, David P. Fee, Paul R. Kemper, Stanley E. Anderson, and Michael T. Bowers, Int. J. Mass Spectrom. 2003, 227, 205-216.
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MALDI-TOF
TOF Mode
m/zErin S. Baker, Jennifer Gidden, David P. Fee, Paul R. Kemper, Stanley E. Anderson, and Michael T. Bowers, Int. J. Mass Spectrom. 2003, 227, 205-216.
TOFDrift Cell
TOF Detector
hν
Source
Quadrupole
Mass Spectrum
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MALDI-TOF
Ion Mobility Mode
TOFDrift Cell
Detector
hν
Source
Quadrupole
Single Conformer
Multiple Conformers
Arrival Time Distributions
Erin S. Baker, Jennifer Gidden, David P. Fee, Paul R. Kemper, Stanley E. Anderson, and Michael T. Bowers, Int. J. Mass Spectrom. 2003, 227, 205-216.
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ESI
ESI IonSource
ESI IonSource
IonFunnel
IonFunnel
DriftCell
DriftCell MSMS DetectorDetector
Ion Funnel
DriftCell
Ion Optics
QuadAnalyzer
DetectorTo PumpTo PumpTo Pump
To Pump
Ion Funnel
DriftCell
Ion Optics
QuadAnalyzer
DetectorTo PumpTo PumpTo Pump
To Pump
Thomas Wyttenbach, Paul R. Kemper, and Michael T. Bowers , Int. J. Mass Spectrom. 2001, 212, 13-23.
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Experiment vs. Theory
Molecular Mechanics/Dynamics
Structures Collision Cross-Sections (σ)
ATDs Mobilities (K) Collision Cross-Sections (σ)
Compare
Experimental Method:
Theoretical Method:
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800 K
0 K
30 ps
Simulated annealing
10 ps
Get 100 structures (0 K)& 100 cross-sections
Tem
pera
ture
Time
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Theoretical Method
Structures Collision Cross-Sections (σ)
Relative Energy (kcal/mol)
-5 0 10 15 20 25220
240
260
280
Cro
ss-S
ectio
n (Å
2 )
5
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DNA Background
• DNA encodes all information necessary to develop from sperm and egg to a life form as complex as the one listening to this talk
• DNA does this by ordering four simple bases on a phosphate backbone
• Watson and Crick discovered the double helix structure of DNA
• The 3 main helix forms are A, B and Z
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Adenine
Guanine
DNA Bases and Backbone
N
N
NHH
N
N
R
N
N
R
O
OCH3 H
Purines Pyrimidines
Cytosine
Thymine
N
N
O
N
H
H
HN
N
R
BaseO
OH
O
O
P
O
BaseOP OO
O O
O
O
BaseOPO OO
HOCH2Base
N
N
R
O
NHH
5΄
3΄
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Watson-Crick Base Pairing Schemes
Adenine – Thymine
2 H-Bonds
N
N
O
NH
H
H
N
N
R
N N
O
N
R
H
H
Guanine – Cytosine
3 H-Bonds
N
N
NHH
N
N
R
N N
O
O
CH3
RH
Base O
OH
O
O
P
O
HOCH2Base
OO
BaseO
OH
O
O
P
O
HOCH2 Base
O O
5΄
5΄
3΄
3΄
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A, Z and B Helix Conformations
A-Form DNARight-handed
~11 base pairs / turn
2.55 Å Axial Rise
B-Form DNA
Right-handed
~10.5 base pairs / turn
3.4 Å Axial Rise
Z-Form DNA
Left-handed
~12 base pairs / turn3.7 Å Axial Rise
http://www.lmb.uni-muenchen.de/groups/Biostruc/chap-08/chap-08-slides.html
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What factors effect helix formation and
Watson-Crick base pairing?
Metals ?
Solvents ?
Size ?
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DuplexesPrior Work
• Observed duplexes in several tetra and hepta nucleotides
• Complimentary pairs had larger dissociation energies than non-complimentary pairs (BIRD)
• Dissociation energies correlate with solution dimerization enthalpies
• Short term molecular dynamics suggest Watson-Crick pairing retained but helix lost.
Paul D. Schnier, John S. Klassen, Eric F. Strittmatter, and Evan R. Williams J. Am.Chem. Soc. 1998, 120, 9605-9613.
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dCG Series• Alternating pyrimidine-purine DNA sequences
form Z-form helices as seen in X-ray crystal structures.
• Sequences Analyzed
dCG 2-mer
dCGCG 4-mer
dCGCGCG 6-mer
dCGCGCGCG 8-mer
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2-mer
dCG
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2-mer MALDI-TOF Mass Spectrum
m/z
200 600400 1200
Inte
nsity
Duplex
Single Strand
[dCG / dCG -H]-
[dCG -H]-
800 1000
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2-mer MALDI ATDs at 300K
Arrival Time (µs)
Cu2+[dCG / dCG-H]-
[dCG / dCG-H]-
700 900 13001100
Na+[dCG / dCG]
[dCG-H]-
1500
σ EXPT = 142, 151 Å2
σ EXPT = 217, 225 Å2
σ EXPT = 228 Å2
σ EXPT = 230, 252 Å2
1 2
M+
M+
WC
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[dCG-H]- Theoretical Structures
Relative Energy (kcal/mol)
Cro
ss S
ectio
n (Å
2 )
135
145
155
165
0-5 10 15 25
Stacked
H-Bonded
140
150
160
175
5
170
20
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[dCG-H]- Single Strand Theoretical Structures
σEXPT = 142, 151 Å2
σTheory = 152 Å2
= Strand
= Guanine (G)
= Cytosine (C)
σEXPT = 142,151 Å2
σTheory = 141 Å2
Jennifer Gidden and Michael T. Bowers, Eur. Phys. J. D 2002, 20, 409-419.
3΄
5΄
5΄
3΄
Stacked H-Bonded
S
H
arrival time
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[dCG / dCG-H]- Duplex Theoretical Structures
σEXPT = 217, 225 Å2
σTheory = 225 Å2
2 H-Bonded Pairs1 H-Bonded Pair
σEXPT = 217, 225 Å2
σTheory = 218 Å2
5΄
3΄
3΄
5΄3΄
5΄
3΄
5΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
1 2
arrival time
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Na+[dCG / dCG] Duplex Theoretical Structures
σEXPT = 228 Å2
σTheory = 231 Å2
5΄
5΄
3΄
3΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
M+
arrival time
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Cu2+[dCG / dCG-H]- Duplex Theoretical Structures
σEXPT = 230, 252 Å2
σTheory(WC) = 249 Å2
σTheory(Na+) = 231 Å2
5΄
5΄
3΄3΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
M+
WC
Watson-Crick pair
Watson-Crick pair
Cu2+ ?
arrival time
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4-mer
dCGCG or d(CG)2
Now only Watson-Crick base pairing will be highlighted
in the theoretical structures
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DNA Backbone Deprotonation
BaseO
OH
O
O
P
O
BaseOP OO
O O
O
O
BaseOPO OO
HOCH2Base
BaseO
OH
O
O
P
O
BaseOP OHO
O OH
O
O
BaseOPO OHO
HOCH2Base
Deprotonate
1
2
3
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4-mer MALDI-TOF Mass Spectrum
m/z1050 16501350 1950
Inte
nsity
Duplex
Single Strand
[d(CG)2 / d(CG)2 -H]-
[d(CG)2 -H]-
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4-mer MALDI-TOF Mass Spectrum and ATDs
m/z1050 16501350 1950
Inte
nsity
Duplex
Single Strand
[d(CG)2 / d(CG)2 -H]-
[d(CG)2 -H]-
ATDs
arrival time
arrival time
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[d(CG)2-H]- Single Strand Theoretical StructuresSite of Deprotonation Insignificant
Deprotonation of 2nd PO4 illustrated below
σEXPT = 212 Å2
σTheory = 213 Å2
3΄
= Strand
= Guanine (G)
= Cytosine (C)
5΄
ATD
arrival time
Forms a simple loop with WC pairing at end
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[d(CG)2-H]- Duplex Theoretical Structures
Starting Helix Duplexes
A-FormσTheory = 409 Å2
Z-FormσTheory = 409 Å2
B-FormσTheory = 391 Å2
5΄ 5΄ 5΄
5΄ 5΄5΄3΄
3΄3΄
3΄
3΄
3΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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[d(CG)2 / d(CG)2-H]- Duplex Theoretical Structures
GlobularσEXPT = 330 Å2
σTheory= 333 Å2
Lowest Energy DuplexStarting Helical Duplex
B-FormσEXPT = 330 Å2
σTheory = 391 Å2
3΄
3΄5΄
5΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
5΄
5΄
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4-mer ESI Mass Spectrum
m/z
Inte
nsity
[Single Strand]- ?
[Duplex]3-
[Single Strand]2-
500 700 900 1100 1300
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4-mer ESI Mass Spectrum and ATD
m/z
Inte
nsity
500 700 900 1100 1300
[Single Strand]2-
[Duplex]3-
ATD
arrival time
[Single Strand]- ?
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[d(CG)2-H]- ESI ATDs at 300K
Arrival Time (µs)
70 eV
30 eV
10 eV
100 eV
500 700 900600 800
Injection Energy
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[d(CG)2-H]- ESI ATDs at 300K
Arrival Time (µs)
70 eV
30 eV
10 eV
100 eV
500 700 900600 800
[triplex]3-
[duplex]2-
[single strand]-
σ EXPT = 423, 326, 208 Å2
Injection Energy
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[d(CG)2-H]- Single Strand Theoretical StructuresSite of Deprotonation Insignificant
Deprotonation of 2nd PO4 illustrated below
σEXPT = 208 Å2
σTheory = 213 Å2
3΄
= Strand
= Guanine (G)
= Cytosine (C)
5΄ ATD
arrival time
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[d(CG)2 / d(CG)2-H]2- Duplex Theoretical Structures
Lowest Energy DuplexStarting Helical Duplex
B-FormσEXPT = 326 Å2
σTheory = 391 Å2
3΄
3΄5΄
5΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
5΄
5΄
GlobularσEXPT = 326 Å2
σTheory = 328 Å2
3΄
arrival time
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[d(CG)2-H / d(CG)2-H / d(CG)2-H]3- Triplex Theoretical Structures
GlobularσEXPT = 423 Å2
σTheory = 424 Å2
Lowest Energy Triplex
= Strand 1= Strand 2
= Guanine (G)= Cytosine (C)
Starting Triplex Structures
3΄ 3΄ 3΄
3΄
3΄
3΄
5΄
5΄
5΄
5΄5΄5΄
1
2
= Strand 3
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[d(CG)2-H / d(CG)2-H / d(CG)2-H]3- Triplex Theoretical Structures
GlobularσEXPT = 423 Å2
σTheory = 424 Å2
Lowest Energy Triplex
= Strand 1= Strand 2
= Guanine (G)= Cytosine (C)
= Strand 3
ATD
arrival time
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-1 Single Strand ?
4-mer ESI Mass Spectrum
m/z
Inte
nsity
-3 Duplex
500 700 900 1100 1300
x20
-H
(Na -2H)-
(3Na -4H)-
(2Na -3H)-
(4Na -5H)-
[Single Strand]2-
[Duplex]3-
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-1 Single Strand ?
4-mer ESI Mass Spectrum
m/z
Inte
nsity
-3 Duplex
500 700 900 1100 1300
x20
1196
1174
12071218
m/z = 11 duplex
1185
[Single Strand]2-
[Duplex]3-
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4-mer ESI Mass Spectrum
m/z
Inte
nsity [Single Strand]-
[Duplex]2-
[Triplex]3-
[Duplex]3-
[Single Strand]2-
500 700 900 1100 1300
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What is the conformation of the 4-mer duplex
in water?
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[d(CG)2 / d(CG)2] Duplex Theoretical Structures in WaterAnnealed 4-mer Helical Duplex
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
= Water
500 K
50 K
30 ps 9 ps
Tem
pera
ture
time4 ps
100 K
Annealing Procedure
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[d(CG)2 / d(CG)2] Duplex Theoretical Structures in WaterRemove Water
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
Na+
Na+
Na+
Na+
Na+
Na+
Coordinate to PO4
Coordinate to PO4
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[d(CG)2 / d(CG)2] Duplex Theoretical Structures in Water
Annealed in Water
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
Annealed without Water 1st Cycle
Helical Globular
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6-mer
dCGCGCG or d(CG)3
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6-mer MALDI-TOF Mass Spectrum
m/z1500 30002500 4000
Inte
nsity
Duplex
Single Strand
35002000
[d(CG)3 / d(CG)3 -H]-
[d(CG)3 -H]-
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6-mer MALDI-TOF Mass Spectrum and ATDs
m/z1500 30002500 4000
Inte
nsity
Duplex
Single Strand
35002000
ATDs
arrival time
arrival time
[d(CG)3 / d(CG)3 -H]-
[d(CG)3 -H]-
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[d(CG)3-H]- Single Strand Theoretical Structures
= Strand
= Guanine (G)
= Cytosine (C) σEXPT = 276 Å2
σTheory = 280 Å2
3΄ 5΄
Forms a simple loop with WC pairing at end
Site of Deprotonation Insignificant Deprotonation of 3rd PO4 illustrated below
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[d(CG)3/ d(CG)3-H]- Duplex Theoretical StructuresStarting Helix Duplexes
A-FormσTheory = 526 Å2
Z-FormσTheory = 534 Å2
B-FormσTheory = 515 Å2
3΄
3΄5΄
5΄
3΄
3΄
3΄
3΄
5΄
5΄ 5΄
5΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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[d(CG)3 / d(CG)3-H]- Duplex Theoretical Structures
GlobularσEXPT = 412 Å2
σTheory= 420Å2
Lowest Energy DuplexStarting Helical Duplex
B-FormσEXPT = 412 Å2
σTheory = 515 Å2
5΄
5΄
3΄
3΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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6-mer ESI Mass Spectrum
m/z
Inte
nsity
[Duplex]3-
[Single Strand]2- ?
550 750 950 1150 1300
[Single Strand]3-
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6-mer ESI Mass Spectrum
m/z
Inte
nsity
[Duplex]3-
550 750 950 1150 1300
x20
902
896
907
913
918
m/z = 5.5 duplex
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6-mer ESI Mass Spectrum
m/z
Inte
nsity
[Duplex]3-
[Single Strand]2-
[Duplex]4-
550 750 950 1150 1300
[Single Strand]3-
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6-mer ESI Mass Spectrum and ATD
m/z
Inte
nsity
[Duplex]3-
550 750 950 1150 1300
[Single Strand]3-
ATD
arrival time
[Single Strand]2-
[Duplex]4-
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[d(CG)3-2H]2- ESI ATDs at 300K
Arrival Time (µs)
50 eV
10 eV
400 650600550500450
Injection Energy
40 eV
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[d(CG)3-2H]2- ESI ATDs at 300K
Arrival Time (µs)
50 eV
10 eV
400 650600550500450
σ EXPT = 418, 282 Å2
Injection Energy
[duplex]4-
[single strand]2-
40 eV
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[d(CG)3-2H]2- Single Strand Theoretical Structures
= Strand
= Guanine (G)
= Cytosine (C) σEXPT = 282 Å2
σTheory = 280 Å2
Site of Deprotonation Insignificant Deprotonation at 2nd and 4th PO4 illustrated below
5΄3΄
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[d(CG)3-2H / d(CG)3-2H]4- Duplex Theoretical Structures
GlobularσEXPT = 418 Å2
σTheory= 420Å2
Lowest Energy DuplexStarting Helical Duplex
B-FormσEXPT = 418 Å2
σTheory = 515 Å2
5΄
5΄
3΄
3΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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8-mer
dCGCGCGCG or d(CG)4
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8-mer MALDI-TOF Mass Spectrum
m/z2000 3000
Inte
nsity
Duplex
Single Strand
4000 5000
[d(CG)4 / d(CG)4 -H]-
[d(CG)4 -H]-
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8-mer MALDI-TOF Mass Spectrum and ATDs
m/z2000 3000
Inte
nsity
Duplex
4000 5000
ATDs
arrival time
arrival time Single Strand
[d(CG)4 / d(CG)4 -H]-
[d(CG)4 -H]-
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[d(CG)4-H]- Single Strand Theoretical Structures
σEXPT = 332 Å2
σTheory = 338 Å2
= Strand
= Guanine (G)
= Cytosine (C)
5΄
3΄
Site of Deprotonation InsignificantDeprotonation of 5th PO4 illustrated below
Forms a simple loop with WC pairing at end
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[d(CG)4 / d(CG)4-H]- Duplex Theoretical Structures
A-FormσTheory = 673 Å2
Z-FormσTheory = 668 Å2
B-FormσTheory = 652 Å2
Starting Helix Duplexes3΄
3΄ 3΄
3΄
3΄
3΄
5΄
5΄
5΄
5΄5΄
5΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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[d(CG)4 / d(CG)4-H]- Duplex Theoretical StructuresLowest Energy DuplexStarting Helical Duplex
B-FormσEXPT = 535 Å2
σTheory = 652 Å2
3΄
3΄
5΄
5΄
GlobularσEXPT = 535 Å2
σTheory= 541Å2
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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8-mer ESI Mass Spectrum
m/z
Inte
nsity
[Single Strand]2- ?
[Single Strand]3-
[Single Strand]4-
[Single Strand-G]3-
550 750 950 1150 12501050850650
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8-mer ESI Mass Spectrum
m/z
Inte
nsity
550 750 950 1150 12501050850650
-H
x20
(Na -2H)-
(2Na -3H)-
(3Na -4H)-
(4Na -5H)-
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8-mer ESI Mass Spectrum
m/z
Inte
nsity
550 750 950 1150 12501050850650
x20
1206
1211
1217
1222 1228
m/z = 5.5 duplex
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8-mer ESI Mass Spectrum
m/z
Inte
nsity
[Duplex]4-
[Single Strand]3-
[Single Strand]4-
550 750 950 1150 12501050850650
[Single Strand-G]3-
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8-mer ESI Mass Spectrum
m/z
Inte
nsity
[Single Strand]3-
[Single Strand]4-
550 750 950 1150 12501050850650
ATD
arrival time
[Duplex]4-
[Single Strand-G]3-
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[d(CG)4-2H]2- ESI ATDs at 300K
Arrival Time (µs)
125 eV
25 eV
Injection Energy
80 eV
450 500 550 600 650 700 750 800
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[d(CG)4-2H]2- ESI ATDs at 300K
Arrival Time (µs)
125 eV
25 eV
σ EXPT = 536, 664 Å2
Injection Energy
[duplex]4-
[duplex]4-
80 eV
450 500 550 600 650 700 750 800
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[d(CG)4-2H / d(CG)4-2H]4- Duplex Theoretical StructuresLowest Energy DuplexStarting Helical Duplex
B-FormσEXPT = 536, 664 Å2
σTheory = 652 Å2
3΄
3΄
5΄
5΄
GlobularσEXPT = 536, 664 Å2
σTheory = 541Å2
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
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Is the second peak helical?
A-FormσTheory = 673 Å2
% error = 1.4%
Z-FormσTheory = 668 Å2
% error = 0.6%
B-FormσTheory = 652 Å2
% error = 2.0%
3΄ 3΄ 3΄
3΄
3΄
3΄
5΄
5΄
5΄
5΄5΄
5΄
= Strand 1= Strand 2= Guanine (G)= Cytosine (C)
Experimentσ = 536, 664 Å2
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dCG Series Summary
1. All single strands fold into loops with Watson-Crick pairing at each end
2. MALDI - all oligonucleotide duplexes are globular(Cu2+ stabilizes WC pairing in 2-mer duplex)
3. ESI - 2,4,6-mer duplexes: globular8-mer duplex: globular and helical structures
4. Water stabilizes the 4-mer
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AcknowledgementsAcknowledgements
Dr. Jennifer GiddenDr. Jennifer Gidden
Erin Shammel BakerErin Shammel Baker
Alessandra FerzocoAlessandra Ferzoco
Dr. Paul KemperDr. Paul Kemper
Dr. Thomas WyttenbachDr. Thomas Wyttenbach
Bowers’ GroupBowers’ Group
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Experimental Setup
Ion Source
Mass Spectrometer
Drift Cell
Mass Analyzer Detector
ArrivalTimeDistributions
single conformer
multiple conformers
EXPT: ATDs collisioncross-sections (Ω)mobilities (K)
THEORY: molecular mechanics structures collisioncross-sections (Ω)
(AMBER)
gate (1-10 µs)
= = CΩtd
lvd = K E
MALDI Time-of-Flight l = 20 cmV = 8-16 V/cmp = ~1.5 torr He
Quadrupole
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http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/GP/GeneticCode.html
University of California, Lawrence Livermore National Laboratory, and the Department of Energy
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http://www.lmb.uni-muenchen.de/groups/Biostruc/chap-08/chap-08-slides.html
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A, Z and B Helix Conformations
A-Form DNARight-handed
~11 base pairs / turn
2.55 Å Rise
B-Form DNA
Right-handed
~10.5 base pairs / turn
3.4 Å Rise
Z-Form DNA
Left-handed
~12 base pairs / turn3.7 Å Rise
http://www.lmb.uni-muenchen.de/groups/Biostruc/chap-08/chap-08-slides.html
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dCG Single Strand Series ATDs at 300K
Arrival Time (µs)
[2mer-H]-
[4mer-H]-
[6mer-H]-
[8mer-H]-
σ EXPT = 142, 151 Å2
σ EXPT = 212 Å2
σ EXPT = 276 Å2
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dCG Duplex Series ATDs at 300K
Arrival Time (µs)
[2mer Duplex]-
σ EXPT = 330 Å2
σ EXPT = 217,225 Å2
σ EXPT = 410 Å2
[4mer Duplex]-
[6mer Duplex]-
[8mer Duplex]-
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500 K
50 K
30 ps
Simulated annealing
9 ps
Tem
pera
ture
time4 ps
100 K