Gas-Phase DNA Helix Conformations

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

Outline

• Experimental Method

• Theoretical Method

• Instrumentation

• DNA Background

• DNA Helix Conformations in Gas-Phase

Drift cell

E

p(He)

IonFelFfriction

v = const.v = K EK = ion mobility

Concepts

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

in out

Drift cell

E

1–5 torr He

Ion mobility method

IonSource

IonSource MS1MS1 Drift

CellDriftCell MS2MS2 DetectorDetector

Ion Mobility Instrumentation

Ion Sources

• Matrix Assisted Laser Desorption Ionization (MALDI)

• Electrospray Ionization (ESI) to MS

to MS

LASER

E

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.

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

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.

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.

Experiment vs. Theory

Molecular Mechanics/Dynamics

Structures Collision Cross-Sections (σ)

ATDs Mobilities (K) Collision Cross-Sections (σ)

Compare

Experimental Method:

Theoretical Method:

800 K

0 K

30 ps

Simulated annealing

10 ps

Get 100 structures (0 K)& 100 cross-sections

Tem

pera

ture

Time

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

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

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΄

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΄

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

What factors effect helix formation and

Watson-Crick base pairing?

Metals ?

Solvents ?

Size ?

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.

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

2-mer

dCG

2-mer MALDI-TOF Mass Spectrum

m/z

200 600400 1200

Inte

nsity

Duplex

Single Strand

[dCG / dCG -H]-

[dCG -H]-

800 1000

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

[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

[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

[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

Na+[dCG / dCG] Duplex Theoretical Structures

σEXPT = 228 Å2

σTheory = 231 Å2

5΄

5΄

3΄

3΄


M+

arrival time

Cu2+[dCG / dCG-H]- Duplex Theoretical Structures

σEXPT = 230, 252 Å2

σTheory(WC) = 249 Å2

σTheory(Na+) = 231 Å2

5΄

5΄

3΄3΄


M+

WC

Watson-Crick pair

Watson-Crick pair

Cu2+ ?

arrival time

4-mer

dCGCG or d(CG)2

Now only Watson-Crick base pairing will be highlighted

in the theoretical structures

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


m/z1050 16501350 1950

Inte

nsity

Duplex

Single Strand

[d(CG)2 / d(CG)2 -H]-

[d(CG)2 -H]-

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

[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

[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΄


[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΄


5΄

5΄

4-mer ESI Mass Spectrum

m/z

Inte

nsity

[Single Strand]- ?

[Duplex]3-

[Single Strand]2-

500 700 900 1100 1300

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]- ?

[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

[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

[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

[d(CG)2 / d(CG)2-H]2- Duplex Theoretical Structures



σTheory = 391 Å2

3΄

3΄5΄

5΄


5΄

5΄


σTheory = 328 Å2

3΄

arrival time

[d(CG)2-H / d(CG)2-H / d(CG)2-H]3- Triplex Theoretical Structures


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

[d(CG)2-H / d(CG)2-H / d(CG)2-H]3- Triplex Theoretical Structures


σTheory = 424 Å2

Lowest Energy Triplex

= Strand 1= Strand 2

= Guanine (G)= Cytosine (C)

= Strand 3

ATD

arrival time

-1 Single Strand ?


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-

-1 Single Strand ?


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-


m/z

Inte

nsity [Single Strand]-

[Duplex]2-

[Triplex]3-

[Duplex]3-

[Single Strand]2-

500 700 900 1100 1300

What is the conformation of the 4-mer duplex

in water?

[d(CG)2 / d(CG)2] Duplex Theoretical Structures in WaterAnnealed 4-mer Helical Duplex


= Water

500 K

50 K

30 ps 9 ps

Tem

pera

ture

time4 ps

100 K

Annealing Procedure

[d(CG)2 / d(CG)2] Duplex Theoretical Structures in WaterRemove Water


Na+

Na+

Na+

Na+

Na+

Na+

Coordinate to PO4

Coordinate to PO4

[d(CG)2 / d(CG)2] Duplex Theoretical Structures in Water

Annealed in Water


Annealed without Water 1st Cycle

Helical Globular

6-mer

dCGCGCG or d(CG)3


m/z1500 30002500 4000

Inte

nsity

Duplex

Single Strand

35002000

[d(CG)3 / d(CG)3 -H]-

[d(CG)3 -H]-


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]-

[d(CG)3-H]- Single Strand Theoretical Structures

= Strand

= Guanine (G)

= Cytosine (C) σEXPT = 276 Å2

σTheory = 280 Å2

3΄ 5΄


Site of Deprotonation Insignificant Deprotonation of 3rd PO4 illustrated below

[d(CG)3/ d(CG)3-H]- Duplex Theoretical StructuresStarting Helix Duplexes




3΄

3΄5΄

5΄

3΄

3΄

3΄

3΄

5΄

5΄ 5΄

5΄




σTheory= 420Å2



σTheory = 515 Å2

5΄

5΄

3΄

3΄



m/z

Inte

nsity

[Duplex]3-

[Single Strand]2- ?

550 750 950 1150 1300

[Single Strand]3-


m/z

Inte

nsity

[Duplex]3-

550 750 950 1150 1300

x20

902

896

907

913

918

m/z = 5.5 duplex


m/z

Inte

nsity

[Duplex]3-

[Single Strand]2-

[Duplex]4-

550 750 950 1150 1300

[Single Strand]3-

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-

[d(CG)3-2H]2- ESI ATDs at 300K

Arrival Time (µs)

50 eV

10 eV

400 650600550500450

Injection Energy

40 eV


Arrival Time (µs)

50 eV

10 eV

400 650600550500450

σ EXPT = 418, 282 Å2

Injection Energy

[duplex]4-

[single strand]2-

40 eV

[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΄

[d(CG)3-2H / d(CG)3-2H]4- Duplex Theoretical Structures


σTheory= 420Å2



σTheory = 515 Å2

5΄

5΄

3΄

3΄


8-mer

dCGCGCGCG or d(CG)4


m/z2000 3000

Inte

nsity

Duplex

Single Strand

4000 5000

[d(CG)4 / d(CG)4 -H]-

[d(CG)4 -H]-


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]-

[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






Starting Helix Duplexes3΄

3΄ 3΄

3΄

3΄

3΄

5΄

5΄

5΄

5΄5΄

5΄


[d(CG)4 / d(CG)4-H]- Duplex Theoretical StructuresLowest Energy DuplexStarting Helical Duplex


σTheory = 652 Å2

3΄

3΄

5΄

5΄


σTheory= 541Å2



m/z

Inte

nsity

[Single Strand]2- ?

[Single Strand]3-

[Single Strand]4-

[Single Strand-G]3-

550 750 950 1150 12501050850650


m/z

Inte

nsity

550 750 950 1150 12501050850650

-H

x20

(Na -2H)-

(2Na -3H)-

(3Na -4H)-

(4Na -5H)-


m/z

Inte

nsity

550 750 950 1150 12501050850650

x20

1206

1211

1217

1222 1228

m/z = 5.5 duplex


m/z

Inte

nsity

[Duplex]4-

[Single Strand]3-

[Single Strand]4-

550 750 950 1150 12501050850650

[Single Strand-G]3-


m/z

Inte

nsity

[Single Strand]3-

[Single Strand]4-

550 750 950 1150 12501050850650

ATD

arrival time

[Duplex]4-

[Single Strand-G]3-


Arrival Time (µs)

125 eV

25 eV

Injection Energy

80 eV

450 500 550 600 650 700 750 800


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

[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


Is the second peak helical?


% error = 1.4%


% error = 0.6%


% error = 2.0%

3΄ 3΄ 3΄

3΄

3΄

3΄

5΄

5΄

5΄

5΄5΄

5΄


Experimentσ = 536, 664 Å2

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

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

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

http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/GP/GeneticCode.html

University of California, Lawrence Livermore National Laboratory, and the Department of Energy

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


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

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]-

500 K

50 K

30 ps

Simulated annealing

9 ps

Tem

pera

ture

time4 ps

100 K

Gas-Phase DNA Helix Conformations

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