Nucleic Acid NMR Part II - University of Georgia

34
Nucleic Acid NMR Part II

Transcript of Nucleic Acid NMR Part II - University of Georgia

Page 1: Nucleic Acid NMR Part II - University of Georgia

Nucleic Acid NMR

Part II

Page 2: Nucleic Acid NMR Part II - University of Georgia

!

"

#

$

%

&

'

O5’

O3’

(3(0

(1(2

(4O4’

nu

cle

otid

e u

nit

α and ζ pose problemsDeterminants of 31P chem shift.

ε and ζ correlate. ζ = -317-1.23 ε

Ranges χ α β γ δ ε ζB-DNA -119 -61 180 57 122 -187 -91Bf-DNA -102 -41 136 38 139 -133 -157Af-DNA -154 -90 -149 47 83 -175 -45

Sanger, Principles of nucleic acid StructuresSpringer 1984

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Backbone Experiments: CT-NOESY, CT-COSY

Bax, A., Tjandra, N., Zhengrong, W., ( 2001). Measurements of 1H-

31P dipolar couplings in a DNA

oligonucleotide by constant time NOESY difference spectroscopy, J. Mol. Biol., 19, 367-270.

ε

Attenuated Scan

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Σ Backbone Experiments

• Z. Wu, N. Tjandra, and A. Bax, Measurement of H3’-31P dipolar couplings in a DNA oligonucleotide byconstant-time NOESY difference spectroscopy, J. Biomol. NMR 19, 367-370 (2001).

• A. Bax, N. Tjandra, W. Zhengrong. Measurements of 1H-31P dipolar couplings in a DNA oligonucleotideby constant time NOESY difference spectroscopy, J. Mol. Biol., 19, 367-270, 91 ( 2001).

• G. M. Clore, E. C. Murphy, A. M. Gronenborn, and A. Bax, Determination of three-bond H3’-31Pcouplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy, J.Mag. Reson. 134, 164-167 (1998).

• H. Schwalbe, W. Samstag, J. W. Engels, W. Bermel, & C. Griesinger, "Determination of 3J(C,P) and3J(H,P) Coupling Constants in Nucleotide Oligomers", J. Biomol. NMR 3, 479-486 (1993).

• BioNMR in Drug Research 2003 Edito: O. ZerbeMethods for the Measurement of Angle Restraints from Scalar, Dipolar Couplings and from Cross-Correlated Relaxation: Application to BiomacromoleculesChapter Author: Christian Griesinger:J-Resolved Constant Time Experiment for the Determination of the Phosphodiester Backbone Angles αand ζ.

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Resonance Assignment DNA/RNA (Homonuclear)

A) Non Exchangeable Protons

•Aromatic Spin Systems NOESY, DQFCOSY, TOCSY

•Sugar Spin Systems DQFCOSY, TOCSY

•Sequential Assignment NOESY, 31P-1H HETCOR

B) Exchangeable Protons 1D, NOESY (11, WG, etc)

C) Correlation of Exchangeable NOESY (excitation sculpting)and Non Exchangeable Protons

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NH

N

O

O

H

H

N

N

NH2

O

H

H

CU

A) Assignment of Non Exchangeable Protons

Base and Sugar

COSY/TOCSY C: H5-H6U: H5-H6

TOCSY A: H8-H2 (H2 are generally difficult to assign)

COSY/TOCSY H1’ -H2’ (H2’’) etc

N

NN

N

H2N

H

H A

J Zhang, A Spring, M W Germann J. Am. Chem. Soc. 131 5380. (2009)

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Resonance Assignment DNA/RNA (Homonuclear)

Sequential Assignment

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7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

NOESY Connectivity (e.g. α C Decamer)

G1-H1’

G1-H8

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7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

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7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

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7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

2'2''

2'2''

2'2''

G

αC

T3'-3'

5'-5'

H

H

H

C G T T A A G C G-5’5’-G C G A A T T G C

αCαCalphaC

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5’- CATGCATG

GTACGTAC – 5’

DNA Miniduplex

Excercise

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31P NMR

Two- and Three-dimensional 31P-driven NMR Procedures for complete assignment of backbone resonances inoligodeoxyribonucleotides. G.W. Kellog and B.I. Schweitzer J. Biomol. NMR 3, 577-595 (1993).

HEHAHA HEHAHA-TOCSY

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31P NMR

ppm

4.04.24.44.64.85.05.2 ppm

!-2.0

!-1.5

!-1.0

!-0.5

1.0

0.5

0.0

P7

P8

P3

P2

P6

P4

P1

P5

P9

ppm

4.04.24.44.64.85.05.2 ppm

!-2.0

-!1.5

!-1.0

-!0.5

1.0

0.5

0.0

P6

P4

P8P2

P9

P1P7P5

P3

ppm

4.04.24.44.64.85.05.2 ppm

!-1.5

!-1.0

!-0.5

1.0

0.5

0.0

P2

P3

P5P6 P7

P1P4

P9P8

AlphaC

3’ 4’ 5’,5’’

;****************************************;mwgcorrpt, AMX version;X-H correlation. H-detected;Sklenar et al., 1986, FEBS, 208, 94-98;****************************************d12=20up2=p1*2

1 ze d11 dhi2 d113 d12 p2 ph0 d2 lo to 3 times l1 d3 (p3 ph2):d d0 (p1 ph1) (p3 ph1):d go=2 ph31 d11 wr #0 if #0 id0 ip2 zd lo to 3 times td1 do exit

ph0=0ph1=0ph2=0 0 2 2ph31=2 2 0 0

;>>>>>>>>>>>>>>>DELAYS;d0 = 3us;d2 = 50ms;d3 = 3us;d11= 30 msec

;>>>>>>>>>>>>>>>PULSES;p1 = 90 deg proton pulse hl1 = 1;p2 = 180 deg proton pulse hl1 = 1;p3 = 90 deg X pulse;>>>>>>>>>>>>>>>LOOP-COUNTER;l1 = loop counter for presaturaton;l1*d2 = relaxation delay (l1=40, d2=50ms >>2s);>>>>>>>>>>>>>>>COMMENTS;rd=pw = 0, nd0 = 2, in0 = 1/(2*SW);ns = 4*n, ds = 4, MC2= TPPI;-----------------------END of PROGRAM---------------

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B) Exchangeable Protons

1D Imino Proton Spectrum

PL

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Assignment of Exchangeable Protons

PL

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Correlation between exchangeableand non-exchangeable protons

N

N

N

N

N

NN

O

O

HH

H

H

H1'

AU

N

N

N

N

NN

ON

NO

H

H

H

H

H

H

H1'

GC

RNA

DNA

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Heteronuclear Methods

Resonance Assignment of RNA/DNA by Heteronuclear NMR13C and 15N correlations

A) Exchangeable Protons 15N-1H HSQC15N edited NOESY HSQC (3D)

B) Non Exchangeable Protons• Base/Sugar 13C-1H HSQC

HCCH -TOCSY HCCH-COSY 2/3D• Base-Sugar HCN, H(CNC)H, H(CN)H 2/3D

• Sequential 13C Edited NOESY-HSQC 3/4DPH, P(C)H, HCP 2/3D

C) Correlation of Exchangeable A, C, G, U, T- specific 2Dand Non Exchangeable Protons 13C Edited NOESY-HSQC 3/4D

D) Base Pairing

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PL

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Non-exchangeable protons: CT-HSQC/HMQCUse Constant time experiments (CC couplings in F1)

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Non-exchangeable protons: HCCH-Type Experiments

2

C1’/H1’

C4’/H4’

C2’/H2’C3’/H3’

C5’/H5’C5’’/H5’’

F1F1

RREIIB-Tr, ~300 uM, 298 K

F1 x F2: correlate a specific sugar 1H to its ownsugar 1H’s and their respective 13C’s.

F3 x F2: Correlate each of its own sugar 1H’s tothe 13C of a specific 1H

HCCH COSYHCCH TOCSY

1H 13C 13C 1HINEPT COSY

RELAYTOCSY

INEPT

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PL

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PL

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PL

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PL

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PL

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Structure Determination:

I) Assignment

II) Local Analysis•glycosidic torsion angle, sugar puckering,backbone conformationbase pairing

III) Global Analysis•sequential, inter strand/cross strand, dipolar coupling

Nucleic Acids have few protons…..•NOE accuracy

> account for spin diffusion•Backbone may be difficult to fully characterize•Dipolar couplings

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What do we know?What do we know?••Distance, Torsion, H-Bond constraintsDistance, Torsion, H-Bond constraintsWhat do we want?What do we want?••Low energy structuresLow energy structures

MethodsMethods••Distance GeometryDistance Geometry••Simulated annealing,Simulated annealing, rMDrMD••Torsion angle dynamics (DYANA)Torsion angle dynamics (DYANA)••Mardigras/IRMA/MorassMardigras/IRMA/Morass

1D NMR!

optimize conditionspH, I, T.

Assignments!spin system!sequential!long range

NOESY, TOCSY, COSY

Distance constraintsTorsion constraints

Distance Geometry/simulated annealing

NOESY, COSY

Initial structure(s)

Use contraints to calculate structure

Identify additional constraints(side chains, additional long range contacts etc)

Reffine structure(s) rMD calculations

Structures

Additional Experiments

Dynamics

Mutants

Interaction with target/drug

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Dipolar Couplings… is the kink real ?

• Dipolar couplings add to J coupling• They show up as a field or alignment media dependence of the coupling• If the overall orientation of the molecule is known the orientation of the vectors can be determined

N

NN

N

NH2

H

HO

HO

HHHH

OH

H

DmaxIS = −

µ0γ IγSh4π 2rIS

3

)1cos3(21 2

max −= θISIS DD

B0

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Aligned with pf1

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αA

NOE RDC + NOE

RMSD (all atoms) 0.66 C3' DG5 1 -- H3' C4' DT 2 -- H4'

C6 DT 2 -- H6 C1' DC 4 -- H1'

C1' ADA 5 -- H1' C4' ADA 5 -- H4' C2 ADA 5 -- H2

C4' DC 6 -- H4' C8 DA 8 -- H8

C1' DC 9 -- H1' C3' DC 9 -- H3' C6 DC 9 -- H6

C1' DG3 10 -- H1' C4' DG3 10 -- H4'

C1' DC5 11 -- H1' C4' DC5 11 -- H4' C1' DT 13 -- H1'

C6 DT 13 -- H6 C4' DC 14 -- H4'

C6 DC 14 -- H6 C8 DG 15 -- H8 C1' DT 16 -- H1'

C1' DG 17 -- H1' C3' DC3 20 -- H3'

C4' DC3 20 -- H4'

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General references, NMR techniques, sample preparation, analysis BioNMR in Drug Research. Edited by Oliver Zerbe, 2002 Wiley Verlag Wijmenga, S. S., Mooren, M. M. W. and Hilbers, C. W. (1993) in Roberts, G. C. K. (ed.) NMR of

Macromolecules; A Practical Approach. Oxford University Press, NY. Zidek L., Stefl R and Sklenar V. (2001) "NMR methodology for the study of nucleic acids"Curr.

Opin. Struct. Biol., 11, 275-28

NMR structure determination: DNA DNA/RNA, pseudorotation analysis, dynamics. See also referenced quoted in the listed papers Altona, C., Francke, R., de Haan, R., Ippel, J. H., Daalmans, G. J., Westra Hoekzema, A. J. A.

and van Wijk, J. (1994) Magn. Reson. Chem., 32, 670-678. Aramini, J. M., Cleaver, S. H., Pon, R. T., Cunningham, R. P. & Germann, M.W: Solution

Structure of a DNA Duplex Containing an a -Anomeric Adenosine: Insights into Substrate Recognition by Endonuclease IV. J. Mol. Biol. (2004), 338, 77-91.

Aramini, J. M., Mujeeb, A., Ulyanov, N. B. & Germann, M. W.: Conformational Dynamics in Mixed a /b- Oligonucleotides Containing Polarity Reversals: A Molecular Dynamics Study using Time-averaged Restraints. J. Biomol. NMR, (2000), 18, 287-303.

Aramini, J. M. & Germann, M. W. NMR solution structure of a DNA/RNA hybrid containing an alpha anomeric thymidine and polarity reversals. Biochemistry, (1999), 38, 15448-15458.

Donders, L. A., de Leeuw, F. A. A. M. and Altona, C. (1989) Magn. Reson. Chem., 27, 556-563. van Wijk, J., Huckriede, B. D., Ippel, J. H. & Altona, C. (1992) Methods Enzymol., 211, 286-306. Bax, A., Lerner, L.. "MEASUREMENT OF H-1-H-1 COUPLING-CONSTANTS IN DNA

FRAGMENTS BY 2D NMR." . J Magn Reson. 79 429 - 438, 1988.. Szyperski, T., Fernández, C., Ono, A., Kainosho, M. and Wüthrich, K. (1998) Measurement of

Deoxyribose 3 JHH Scalar Couplings Reveals Protein-Binding Induced Changes in the Sugar Puckers of the DNA. J. Am. Chem. Soc. 120, 821- 822

Iwahara J, Wojciak JM, Clubb RT. (2001), An efficient NMR experiment for analyzing sugar-puckering in unlabeled DNA: application to the 26-kDa dead ringer-DNA complex. J Magn Reson. 2001, 153, 262

Multinuclear experiments, DNA/RNA Pardi, A. and Nikonowicz, E.P. (1992) Simple procedure for resonance assignment of the sugar

protons in 13C labeled RNA J. Am. Chem. Soc., 114, 9202–9203 Sklénar, V., Miyashiro, H., Zon, G., Miles, H.T., Bax, A. (1986) Assignment of the 31P and 1H

resonances in oligonucleotides by two-dimensional NMR spectroscopy FEBS Lett., 208, 94–9

Varani, G., Aboul-ela, F., Allain, F., Gubser, C.C. (1995) Novel three-dimensional 1H–13C–31P triple resonance experiments for sequential backbone correlations in nucleic acids J. Biomol. NMR, 5, 315–3

Legault, P., Farmer, B.T. II , Mueller, L. and Pardi, A. (1994) Through-bond correlation of adenine protons in a 13C-labeled ribozyme. J. Am. Chem. Soc., 116, 2203-2204

Marino, J.P, Prestegard, J.H. & Crothers, D.M. (1994) Correlation of adenine H2/H8 resonances in uniformly 13C labeled RNAs by 2d HCCH-TOCSY: a new tool for 1H assignment. J. Am. Chem. Soc., 116, 2205-2206

Sklenár, V., Peterson, R.D., Rejante, M.R., Wang, E. & Feigon, J. (1993) Two-dimensional triple-resonance HCNCH experiment for direct correlation of ribose H1 and Base H8, H6 protons in 13C, 15N-labeled RNA oligonucleotides. J. Am. Chem. Soc., 115, 12181-12182

Sklenár, V. Peterson, R.D., Rejante, M.R. & Feigon, J. (1994) Correlation of nucleotide base and sugar protons in 15N labeled HIV RNA oligonucleotide by 1H-15N HSQC experiments. J. Biomol. NMR, 4, 117-122

P Schmieder, J H Ippel, H van den Elst, G A van der Marel, J H van Boom, C Altona, and H Kessler (1992) Heteronuclear NMR of DNA with the heteronucleus in natural abundance: facilitated assignment and extraction of coupling constants. Nucleic Acids Res. 25; 4747–4751.

H. Schwalbe, J. P. Marino, G. C. King, R. Wechselberger, W. Bermel, and C. Griesinger (1994) "Determination of a complete set of coupling constants in 13C-labeled oligonucleotides", J. Biomol. NMR 4, 631-644

Trantirek L., Stefl R., Masse J.E., Feigon J. and Sklenar V. (2002)"Determination of the glycosidic torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1'" J. Biomol. NMR., 23(1):1-12

Szyperski, T., Ono, A., Fernández, C., Iwai, H., Tate, S., Wüthrich, K. and Kainosho, M. (1997) Measurement of 3JC2'P Scalar Couplings in a 17 kDa Protein Complex with 13 C,15N-Labeled DNA Distinguishes the B I and BII Phosphate Conformations of the DNA. J. Am. Chem. Soc. 119, 9901 -990

Szyperski, T., Fernandez, C., Ono, A., Wüthrich, K. and Kainosho, M. (1999) The { 31P}-Spin-echo-difference Constant-time [ 13C,1 H]-HMQC Experiment for Simultaneous Determination of 3 JH3'P and 3JC4'P in Nucleic Acids and their Protein Complexes. J. Magn. Reson. 140, 491 -494.

H. Schwalbe, W. Samstag, J. W. Engels, W. Bermel, and C. Griesinger, (1993) "Determination of 3J(C,P) and 3J(H,P) Coupling Constants in Nucleotide Oligomers", J. Biomol. NMR 3, 479-486

C. Richter, B. Reif, K. Wörner, S. Quant, J. W. Engels, C. Griesinger, and H. Schwalbe (1998) "New Experiment for the Measurement of 3J(C,P) Coupling Constants including 3J(C4'i,Pi) and 3J(C4'i,P i+1) coupling constants in Oligonucleotides" J. Biomol. NMR 12, 223-23