Gas-Phase DNA Helix Conformations

Transcription

1 Gas-Phase DNA Helix Conformations Erin Shammel Baker, Jennifer Gidden, Alessandra Ferzoco, Thomas Wyttenbach and Michael Bowers

2 utline Experimental Method Theoretical Method Instrumentation DNA Background DNA Helix Conformations in Gas-Phase

3 Concepts E F friction Ion F el p(he) v = const. v = K E Drift cell K = ion mobility

4 K = f (T, p, q, µ, σ) T = temperature p = pressure q = ion charge µ = reduced mass K = ion mobility σ = collision cross section σ = f ( ) He ion interaction Ion shape

5 Ion mobility method E in out 1 5 torr He Drift cell

6 Ion Mobility Instrumentation Ion Source MS1 Drift Cell MS2 Detector

7 Ion Sources Matrix Assisted Laser Desorption Ionization (MALDI) to MS LASER Electrospray Ionization (ESI) E to MS

8 MALDI-TF hν Source TF Detector TF Quadrupole Drift Cell Glass l = 20 cm p = ~1.5 torr He Erin S. Baker, Jennifer Gidden, David P. Fee, Paul R. Kemper, Stanley E. Anderson, and Michael T. Bowers, Int. J. Mass Spectrom. 2003, 227,

9 MALDI-TF hν TF Mode Source TF Detector TF Mass Spectrum Drift Cell Quadrupole m/z Erin S. Baker, Jennifer Gidden, David P. Fee, Paul R. Kemper, Stanley E. Anderson, and Michael T. Bowers, Int. J. Mass Spectrom. 2003, 227,

10 MALDI-TF hν Ion Mobility Mode TF Drift Cell Quadrupole Detector Source Arrival Time Distributions Single Conformer Multiple Conformers Erin S. Baker, Jennifer Gidden, David P. Fee, Paul R. Kemper, Stanley E. Anderson, and Michael T. Bowers, Int. J. Mass Spectrom. 2003, 227,

11 ESI ESI Ion Source Ion Funnel Drift Cell MS Detector To Pump Drift Cell Quad Analyzer Ion Funnel To Pump Ion ptics To Pump To Pump Detector Thomas Wyttenbach, Paul R. Kemper, and Michael T. Bowers, Int. J. Mass Spectrom. 2001, 212,

12 Experiment vs. Theory Experimental Method: ATDs Mobilities (K) Collision Cross-Sections (σ) Compare Theoretical Method: Molecular Mechanics/Dynamics Structures Collision Cross-Sections (σ)

13 800 K Simulated annealing Temperature 0 K Time 30 ps Get 100 structures (0 K) & 100 cross-sections 10 ps

14 Theoretical Method Structures Collision Cross-Sections (σ) 280 Cross-Section (Å 2 ) Relative Energy (kcal/mol)

15 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

16 Adenine Guanine DNA Bases and Backbone N N N H H N N R N N R C H 3 H Purines Pyrimidines Cytosine Thymine N N N H H H N N R Base H P Base P Base P HCH 2 Base N N R N H H

17 Watson-Crick Base Pairing Schemes CH 3 H N H N H N H N N R HCH 2 Base Base P N R N Adenine Thymine 2 H-Bonds P Base Base HCH 2 N H N H N H N N R H N R H N N H Guanine Cytosine 3 H-Bonds

18 A, Z and B Helix Conformations A-Form DNA Z-Form DNA Right-handed Left-handed ~11 base pairs / turn ~12 base pairs / turn 2.55 Å Axial Rise 3.7 Å Axial Rise B-Form DNA Right-handed ~10.5 base pairs / turn 3.4 Å Axial Rise

19 What factors effect helix formation and Watson-Crick base pairing? Metals? Solvents? Size?

20 Duplexes Prior Work Paul D. Schnier, John S. Klassen, Eric F. Strittmatter, and Evan R. Williams J. Am.Chem. Soc. 1998, 120, bserved 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.

21 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

22 2-mer dcg

23 2-mer MALDI-TF Mass Spectrum Intensity Single Strand [dcg -H] - Duplex [dcg / dcg -H] m/z

24 2-mer MALDI ATDs at 300K [dcg-h] - σ EXPT = 142, 151 Å [dcg / dcg-h] - σ EXPT = 217, 225 Å 2 Na + [dcg / dcg] σ EXPT = 228 Å 2 M + WC Cu 2+ [dcg / dcg-h] - σ EXPT = 230, 252 Å 2 M Arrival Time (µs) 1500

25 [dcg-h] - Theoretical Structures Cross Section (Å 2 ) H-Bonded 140 Stacked Relative Energy (kcal/mol)

26 [dcg-h] - Single Strand Theoretical Structures Stacked H-Bonded σ EXPT = 142,151 Å2 σ Theory = 141 Å 2 S H σ EXPT = 142, 151 Å2 σ Theory = 152 Å 2 = Strand = Guanine (G) = Cytosine (C) arrival time Jennifer Gidden and Michael T. Bowers, Eur. Phys. J. D 2002, 20,

27 [dcg / dcg-h] - Duplex Theoretical Structures 1 H-Bonded Pair 2 H-Bonded Pairs σ EXPT = 217, 225 Å2 σ Theory = 218 Å σ EXPT = 217, 225 Å2 σ Theory = 225 Å 2 = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) arrival time

28 Na + [dcg / dcg] Duplex Theoretical Structures = Strand 1 σ EXPT = 228 Å 2 σ Theory = 231 Å 2 M + = Strand 2 = Guanine (G) = Cytosine (C) arrival time

29 Cu 2+ [dcg / dcg-h] - Duplex Theoretical Structures Cu 2+? Watson-Crick pair Watson-Crick pair = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) σ EXPT = 230, 252 Å 2 σ Theory(WC) = 249 Å 2 σ Theory(Na+) = 231 Å 2 WC M + arrival time

30 4-mer dcgcg or d(cg) 2 Now only Watson-Crick base pairing will be highlighted in the theoretical structures

31 DNA Backbone Deprotonation Base H P Base P Base P HCH 2 Base Base H P Base P H H Base P H HCH 2 Base Deprotonate 1 2 3

32 4-mer MALDI-TF Mass Spectrum Intensity Single Strand [d(cg) 2 -H] - Duplex [d(cg) 2 / d(cg) 2 -H] m/z

33 4-mer MALDI-TF Mass Spectrum and ATDs ATDs arrival time Intensity Single Strand [d(cg) 2 -H] - arrival time Duplex [d(cg) 2 / d(cg) 2 -H] m/z

34 [d(cg) 2 -H] - Single Strand Theoretical Structures Site of Deprotonation Insignificant Deprotonation of 2 nd P 4 illustrated below Forms a simple loop with WC pairing at end ATD = Strand = Guanine (G) = Cytosine (C) σ EXPT = 212 Å2 σ Theory = 213 Å 2 arrival time

35 [d(cg) 2 -H] - Duplex Theoretical Structures Starting Helix Duplexes A-Form σ Theory = 409 Å 2 B-Form σ Theory = 391 Å 2 Z-Form σ Theory = 409 Å 2 = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C)

36 [d(cg) 2 / d(cg) 2 -H] - Duplex Theoretical Structures Starting Helical Duplex Lowest Energy Duplex = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) B-Form σ EXPT = 330 Å2 σ Theory = 391 Å 2 Globular σ EXPT = 330 Å 2 σ Theory = 333 Å 2

37 4-mer ESI Mass Spectrum [Single Strand] 2- Intensity [Single Strand] -? [Duplex] m/z

38 4-mer ESI Mass Spectrum and ATD [Single Strand] 2- ATD Intensity arrival time [Single Strand] -? [Duplex] m/z

39 Injection Energy [d(cg) 2 -H] - ESI ATDs at 300K 100 ev 70 ev 30 ev 10 ev Arrival Time (µs)

40 Injection Energy [d(cg) 2 -H] - ESI ATDs at 300K [single strand] ev [triplex] 3- [duplex] 2- σ EXPT = 423, 326, 208 Å 2 70 ev 30 ev 10 ev Arrival Time (µs)

41 [d(cg) 2 -H] - Single Strand Theoretical Structures Site of Deprotonation Insignificant Deprotonation of 2 nd P 4 illustrated below ATD = Strand = Guanine (G) = Cytosine (C) σ EXPT = 208 Å2 σ Theory = 213 Å 2 arrival time

42 [d(cg) 2 / d(cg) 2 -H] 2- Duplex Theoretical Structures Starting Helical Duplex Lowest Energy Duplex B-Form σ EXPT = 326 Å2 = Strand 1 σ Theory = 391 Å 2 = Strand 2 = Guanine (G) = Cytosine (C) arrival time Globular σ EXPT = 326 Å 2 σ Theory = 328 Å 2

43 [d(cg) 2 -H / d(cg) 2 -H / d(cg) 2 -H] 3- Triplex Theoretical Structures Starting Triplex Structures Lowest Energy Triplex 1 2 = Strand 1 = Strand 2 = Strand 3 = Guanine (G) = Cytosine (C) Globular σ EXPT = 423 Å 2 σ Theory = 424 Å 2

44 [d(cg) 2 -H / d(cg) 2 -H / d(cg) 2 -H] 3- Triplex Theoretical Structures ATD Lowest Energy Triplex arrival time = Strand 1 = Strand 2 = Strand 3 = Guanine (G) = Cytosine (C) Globular σ EXPT = 423 Å2 σ Theory = 424 Å 2

45 4-mer ESI Mass Spectrum [Single Strand] 2- (3Na -4H) - (2Na -3H) - (4Na -5H) - (Na -2H) - Intensity -H -1 Single Strand? -3 [Duplex] 3- x m/z

46 4-mer ESI Mass Spectrum [Single Strand] 2- m/z = 11 duplex Intensity Single Strand? -3 [Duplex] 3- x m/z

47 4-mer ESI Mass Spectrum [Single Strand] 2- Intensity [Single Strand] - [Duplex] 2- [Triplex] 3- [Duplex] m/z

48 What is the conformation of the 4-mer duplex in water?

49 [d(cg) 2 / d(cg) 2 ] Duplex Theoretical Structures in Water Annealed 4-mer Helical Duplex 500 K Annealing Procedure = Water = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) Temperature 100 K 50 K 30 ps 4 ps time 9 ps

50 Na + Na + [d(cg) 2 / d(cg) 2 ] Duplex Theoretical Structures in Water Remove Water Coordinate to P 4 Na + Na + = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) Na + Na+ Coordinate to P 4

51 [d(cg) 2 / d(cg) 2 ] Duplex Theoretical Structures in Water Annealed in Water Annealed without Water 1 st Cycle Helical Globular = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C)

52 6-mer dcgcgcg or d(cg) 3

53 6-mer MALDI-TF Mass Spectrum Intensity Single Strand [d(cg) 3 -H] - Duplex [d(cg) 3 / d(cg) 3 -H] m/z

54 6-mer MALDI-TF Mass Spectrum and ATDs ATDs Intensity arrival time Single Strand [d(cg) 3 -H] - arrival time Duplex [d(cg) 3 / d(cg) 3 -H] m/z

55 [d(cg) 3 -H] - Single Strand Theoretical Structures Site of Deprotonation Insignificant Deprotonation of 3 rd P 4 illustrated below Forms a simple loop with WC pairing at end = Strand = Guanine (G) = Cytosine (C) σ EXPT = 276 Å2 σ Theory = 280 Å 2

56 [d(cg) 3 / d(cg) 3 -H] - Duplex Theoretical Structures Starting Helix Duplexes A-Form σ Theory = 526 Å 2 B-Form σ Theory = 515 Å 2 Z-Form σ Theory = 534 Å 2 = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C)

57 [d(cg) 3 / d(cg) 3 -H] - Duplex Theoretical Structures Starting Helical Duplex Lowest Energy Duplex = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) B-Form σ EXPT = 412 Å2 σ Theory = 515 Å 2 Globular σ EXPT = 412 Å 2 σ Theory = 420Å 2

58 6-mer ESI Mass Spectrum [Single Strand] 3- Intensity [Single Strand] 2-? [Duplex] m/z

59 6-mer ESI Mass Spectrum 896 m/z = 5.5 duplex Intensity 913 x20 [Duplex] m/z

60 6-mer ESI Mass Spectrum [Single Strand] 3- Intensity [Single Strand] 2- [Duplex] 4- [Duplex] m/z

61 [Single Strand] 3-6-mer ESI Mass Spectrum and ATD ATD Intensity arrival time [Single Strand] 2- [Duplex] 4- [Duplex] m/z

62 Injection Energy [d(cg) 3-2H] 2- ESI ATDs at 300K 50 ev 40 ev 10 ev Arrival Time (µs)

63 Injection Energy [d(cg) 3-2H] 2- ESI ATDs at 300K 50 ev 40 ev [single strand] 2-10 ev [duplex] 4- σ EXPT = 418, 282 Å Arrival Time (µs)

64 [d(cg) 3-2H] 2- Single Strand Theoretical Structures Site of Deprotonation Insignificant Deprotonation at 2 nd and 4 th P 4 illustrated below = Strand = Guanine (G) = Cytosine (C) σ EXPT = 282 Å2 σ Theory = 280 Å 2

65 [d(cg) 3-2H / d(cg) 3-2H] 4- Duplex Theoretical Structures Starting Helical Duplex Lowest Energy Duplex = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) B-Form σ EXPT = 418 Å2 σ Theory = 515 Å 2 Globular σ EXPT = 418 Å 2 σ Theory = 420Å 2

66 8-mer dcgcgcgcg or d(cg) 4

67 8-mer MALDI-TF Mass Spectrum Intensity Single Strand [d(cg) 4 -H] - Duplex [d(cg) 4 / d(cg) 4 -H] m/z

68 8-mer MALDI-TF Mass Spectrum and ATDs ATDs Intensity arrival time Single Strand [d(cg) 4 -H] - arrival time Duplex [d(cg) 4 / d(cg) 4 -H] m/z

69 [d(cg) 4 -H] - Single Strand Theoretical Structures Site of Deprotonation Insignificant Deprotonation of 5 th P 4 illustrated below Forms a simple loop with WC pairing at end = Strand = Guanine (G) = Cytosine (C) σ EXPT = 332 Å 2 σ Theory = 338 Å 2

70 [d(cg) 4 / d(cg) 4 -H] - Duplex Theoretical Structures Starting Helix Duplexes A-Form σ Theory = 673 Å 2 B-Form σ Theory = 652 Å 2 Z-Form σ Theory = 668 Å 2 = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C)

71 [d(cg) 4 / d(cg) 4 -H] - Duplex Theoretical Structures Starting Helical Duplex Lowest Energy Duplex = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) B-Form σ EXPT = 535 Å2 σ Theory = 652 Å 2 Globular σ EXPT = 535 Å 2 σ Theory = 541Å 2

72 8-mer ESI Mass Spectrum [Single Strand] 4- [Single Strand] 3- Intensity [Single Strand-G] 3- [Single Strand] 2-? m/z

73 8-mer ESI Mass Spectrum (Na -2H) - -H (2Na -3H) - Intensity (3Na -4H) - (4Na -5H) - x m/z

74 8-mer ESI Mass Spectrum 1211 m/z = 5.5 duplex Intensity x m/z

75 8-mer ESI Mass Spectrum [Single Strand] 4- [Single Strand] 3- Intensity [Single Strand-G] 3- [Duplex] m/z

76 8-mer ESI Mass Spectrum [Single Strand] 4- [Single Strand] 3- ATD Intensity [Single Strand-G] 3- arrival time [Duplex] m/z

77 Injection Energy [d(cg) 4-2H] 2- ESI ATDs at 300K 125 ev 80 ev 25 ev Arrival Time (µs)

78 Injection Energy [d(cg) 4-2H] 2- ESI ATDs at 300K 125 ev 80 ev [duplex] 4-25 ev [duplex] 4- σ EXPT = 536, 664 Å Arrival Time (µs)

79 [d(cg) 4-2H / d(cg) 4-2H] 4- Duplex Theoretical Structures Starting Helical Duplex Lowest Energy Duplex = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) B-Form σ EXPT = 536, 664 Å2 σ Theory = 652 Å 2 Globular σ EXPT = 536, 664 Å 2 σ Theory = 541Å 2

80 Is the second peak helical? A-Form σ Theory = 673 Å 2 % error = 1.4% B-Form σ Theory = 652 Å 2 % error = 2.0% Z-Form σ Theory = 668 Å 2 % error = 0.6% = Strand 1 = Strand 2 = Guanine (G) = Cytosine (C) Experiment σ = 536, 664 Å 2

81 dcg Series Summary 1. All single strands fold into loops with Watson-Crick pairing at each end 2. MALDI - all oligonucleotide duplexes are globular (Cu 2+ stabilizes WC pairing in 2-mer duplex) 3. ESI - 2,4,6-mer duplexes: globular 8-mer duplex: globular and helical structures 4. Water stabilizes the 4-mer

82 Acknowledgements Dr. Jennifer Gidden Erin Shammel Baker Alessandra Ferzoco Dr. Paul Kemper Dr. Thomas Wyttenbach Bowers Group

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85 Experimental Setup gate (1-10 µs) Ion Source Mass Spectrometer Drift Cell Mass Analyzer Detector Arrival Time Distributions MALDI Time-of-Flight l = 20 cm V = 8-16 V/cm p = ~1.5 torr He Quadrupole single conformer multiple conformers EXPT: ATDs mobilities (K) l v = = C d = t K E d Ω THERY: molecular mechanics structures (AMBER) collision cross-sections (Ω) collision cross-sections (Ω)

86

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

88

89 A, Z and B Helix Conformations A-Form DNA Z-Form DNA Right-handed Left-handed ~11 base pairs / turn ~12 base pairs / turn 2.55 Å Rise Å Rise B-Form DNA Right-handed ~10.5 base pairs / turn 3.4 Å Rise

90 dcg Single Strand Series ATDs at 300K [2mer-H] - σ EXPT = 142, 151 Å 2 [4mer-H] - σ EXPT = 212 Å 2 [6mer-H] - σ EXPT = 276 Å 2 [8mer-H] - Arrival Time (µs)

91 dcg Duplex Series ATDs at 300K [2mer Duplex] - σ EXPT = 217,225 Å 2 [4mer Duplex] - σ EXPT = 330 Å 2 [6mer Duplex] - σ EXPT = 410 Å 2 [8mer Duplex] - Arrival Time (µs)

92 Simulated annealing 500 K Temperature 100 K 50 K 4 ps 30 ps time 9 ps