Nucleic Acid NMR Part II - University of Georgia
Transcript of Nucleic Acid NMR Part II - University of Georgia
Nucleic Acid NMR
Part II
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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
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
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.
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
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)
Resonance Assignment DNA/RNA (Homonuclear)
Sequential Assignment
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
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
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
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
5’- CATGCATG
GTACGTAC – 5’
DNA Miniduplex
Excercise
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
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---------------
B) Exchangeable Protons
1D Imino Proton Spectrum
PL
Assignment of Exchangeable Protons
PL
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
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
PL
Non-exchangeable protons: CT-HSQC/HMQCUse Constant time experiments (CC couplings in F1)
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
PL
PL
PL
PL
PL
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
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
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
Iθ
Aligned with pf1
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'
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