Nucleic Acid NMR. Part II

Transcription

1 ucleic Acid MR Part II

2 nucleotide unit " O3! $ # % (4 O4 (3 (0 (1 (2 ' α and ζ pose problems Determinants of 31 P chem shift. ε and ζ correlate. ζ = ε & O5 Ranges χ α β γ δ ε ζ B-DA Bf-DA Af-DA Sanger, Principles of nucleic acid Springer 1984

3 Backbone Experiments: CT-OESY, CT-COSY ε Attenuated Scan Bax, A., Tjandra,., Zhengrong, W., ( 2001). Measurements of 1-31P dipolar couplings in a DA oligonucleotide by constant time OESY difference spectroscopy, J. Mol. Biol., 19,

4 Σ Backbone Experiments Z. Wu,. Tjandra, and A. Bax, Measurement of 3-31 P dipolar couplings in a DA oligonucleotide by constant-time OESY difference spectroscopy, J. Biomol. MR 19, (2001). A. Bax,. Tjandra, W. Zhengrong. Measurements of 1-31 P dipolar couplings in a DA oligonucleotide by constant time OESY difference spectroscopy, J. Mol. Biol., 19, , 91 ( 2001). G. M. Clore, E. C. Murphy, A. M. Gronenborn, and A. Bax, Determination of three-bond 3-31 P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy, J. Mag. Reson. 134, (1998).. Schwalbe, W. Samstag, J. W. Engels, W. Bermel, & C. Griesinger, "Determination of 3 J(C,P) and 3 J(,P) Coupling Constants in ucleotide Oligomers", J. Biomol. MR 3, (1993). BioMR in Drug Research 2003 Edito: O. Zerbe Methods 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 α and ζ.

5 Resonance Assignment DA/RA (omonuclear) A) on Exchangeable Protons Aromatic Spin Systems OESY, DQFCOSY, TOCSY Sugar Spin Systems DQFCOSY, TOCSY Sequential Assignment OESY, 31 P- 1 ETCOR B) Exchangeable Protons 1D, OESY (11, WG, etc) C) Correlation of Exchangeable OESY (excitation sculpting) and on Exchangeable Protons

6 A) Assignment of on Exchangeable Protons Base and Sugar COSY/TOCSY C: 5-6 U: 5-6 TOCSY A: 8-2 (2 are generally difficult to assign) COSY/TOCSY 1-2 (2 ) etc O 2 2 U O C O A J Zhang, A Spring, M W Germann J. Am. Chem. Soc

7 Resonance Assignment DA/RA (omonuclear) Sequential Assignment

8 OESY Connectivity (e.g. α C Decamer) ppm T6 C2 T7 C10 αc8 G G3 G9 G1 A5 A ppm G1-1

9 ppm T6 C2 T7 C10 αc G3 G9 G1 A5 A ppm

10 ppm T6 C2 T7 C10 αc G3 G9 G1 A5 A ppm

11 alphac 5 -G C C G G A T A T T A T α C G C α C A G C G-5 ppm T C2 T7 C10 αc8 T αc 2'2'' 2'2'' 3' G3 G9 G1 G 5'-5' 8.2 A5 A4 2'2'' ppm

12 DA Miniduplex 5 - CATGCATG GTACGTAC 5 Excercise

13 31P MR EAA EAA-TOCSY Two- and Three-dimensional 31P-driven MR Procedures for complete assignment of backbone resonances in oligodeoxyribonucleotides. G.W. Kellog and B.I. Schweitzer J. Biomol. MR 3, (1993).

14 31 P MR ppm!-2.0!-1.5!-1.0! ,5 3 4 AlphaC P7 P3 P6 P5!- -!- - P4 P1 P2p P9!-!- ;**************************************** ;mwgcorrpt, AMX version ;X- correlation. -detected ;Sklenar et al., 1986, FEBS, 208, ;**************************************** d12=20u p2=p1*2 1 ze d11 dhi 2 d11 3 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=0 ph1=0 ph2= ph31= ;>>>>>>>>>>>>>>>DELAYS ;d0 = 3us ;d2 = 50ms ;d3 = 3us ;d11= 30 msec ppm P8!- ;>>>>>>>>>>>>>>>PULSES ;p1 = 90 deg proton pulse hl1 = 1 ;p2 = 180 deg proton pulse hl1 = 1 ;p3 = 90 deg X pulse ;>>>>>>>>>>>>>>>LOOP-COUTER ;l1 = loop counter for presaturaton ;l1*d2 = relaxation delay (l1=40, d2=50ms >>2s) ;>>>>>>>>>>>>>>>COMMETS ;rd=pw = 0, nd0 = 2, in0 = 1/(2*SW) ;ns = 4*n, ds = 4, MC2= TPPI ; ED of PROGRAM

15 B) Exchangeable Protons 1D Imino Proton Spectrum

16 Assignment of Exchangeable Protons

17 Correlation between exchangeable and non-exchangeable protons A O U R O 1' 1' G O O C D

18 eteronuclear Methods Resonance Assignment of RA/DA by eteronuclear MR 13 C and 15 correlations A) Exchangeable Protons 15-1 SQC 15 edited OESY SQC (3D) B) on Exchangeable Protons Base/Sugar 13 C- 1 SQC CC -TOCSY CC-COSY 2/3D Base-Sugar C, (CC), (C) 2/3D Sequential 13 C Edited OESY-SQC 3/4D P, P(C), CP 2/3D C) Correlation of Exchangeable A, C, G, U, T- specific 2D and on Exchangeable Protons 13 C Edited OESY-SQC 3/4D D) Base Pairing

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20 on-exchangeable protons: CT-SQC/MQC Use Constant time experiments (CC couplings in F1)

21 on-exchangeable protons: CC-Type Experiment CC COSY CC TOCSY 1 13 C 13 C 1 F1 x F2: correlate a specific sugar 1 to its own sugar 1 s and their respective 13 C s. RREIIB-Tr, ~300 um, 298 K F1 F1 2 C2 /2 C3 /3 C5 /5 C5 /5 IEPT COSY RELAY TOCSY IEPT C4 /4 C1 /1 F3 x F2: Correlate each of its own sugar 1 s to the 13 C of a specific 1

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29 Structure Determination: I) Assignment II) III) Local Analysis glycosidic torsion angle, sugar puckering,backbone conformation base pairing Global Analysis sequential, inter strand/cross strand, dipolar coupling ucleic Acids have few protons.. OE accuracy > account for spin diffusion Backbone may be difficult to fully characterize Dipolar couplings

30 What do we know? Distance, Torsion, -Bond constraints What do we want? Low energy structures Methods Distance Geometry Simulated annealing, rmd Torsion angle dynamics (DYAA) Mardigras/IRMA/Morass optimize conditions p, I, T. Assignments!spin system!sequential!long range Distance constraints Torsion constraints Distance Geometry/ simulated annealing 1D MR! OESY, TOCSY, COSY OESY, COSY Use contraints to calculate structure Initial structure(s) Identify additional constraints (side chains, additional long range contacts etc) Reffine structure(s) rmd calculations Structures Additional Experiments Dynamics Mutants Interaction with target/drug

31 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 IS D max D IS = µ 0γ I γ S h 4π 2 r IS IS = Dmax (3cos θ 1) 2 B 0 θ 2 O O O

32 Aligned with pf1

33 RMSD (all atoms) 0.66 OE αa RDC + OE C3' DG ' C4' DT ' C6 DT C1' DC ' C1' ADA ' C4' ADA ' C2 ADA C4' DC ' C8 DA C1' DC ' C3' DC ' C6 DC C1' DG ' C4' DG ' C1' DC ' C4' DC ' C1' DT ' C6 DT C4' DC ' C6 DC C8 DG C1' DT ' C1' DG ' C3' DC ' C4' DC '

34 General references, MR techniques, sample preparation, analysis BioMR in Drug Research. Edited by Oliver Zerbe, 2002 Wiley Verlag Wijmenga, S. S., Mooren, M. M. W. and ilbers, C. W. (1993) in Roberts, G. C. K. (ed.) MR of Macromolecules; A Practical Approach. Oxford University Press, Y. Zidek L., Stefl R and Sklenar V. (2001) "MR methodology for the study of nucleic acids"curr. Opin. Struct. Biol., 11, MR structure determination: DA DA/RA, pseudorotation analysis, dynamics. See also referenced quoted in the listed papers Altona, C., Francke, R., de aan, R., Ippel, J.., Daalmans, G. J., Westra oekzema, A. J. A. and van Wijk, J. (1994) Magn. Reson. Chem., 32, Aramini, J. M., Cleaver, S.., Pon, R. T., Cunningham, R. P. & Germann, M.W: Solution Structure of a DA Duplex Containing an a -Anomeric Adenosine: Insights into Substrate Recognition by Endonuclease IV. J. Mol. Biol. (2004), 338, Aramini, J. M., Mujeeb, A., Ulyanov,. B. & Germann, M. W.: Conformational Dynamics in Mixed a /b- Oligonucleotides Containing Polarity Reversals: A Molecular Dynamics Study using Time-averaged Restraints. J. Biomol. MR, (2000), 18, Aramini, J. M. & Germann, M. W. MR solution structure of a DA/RA hybrid containing an alpha anomeric thymidine and polarity reversals. Biochemistry, (1999), 38, Donders, L. A., de Leeuw, F. A. A. M. and Altona, C. (1989) Magn. Reson. Chem., 27, van Wijk, J., uckriede, B. D., Ippel, J.. & Altona, C. (1992) Methods Enzymol., 211, Bax, A., Lerner, L.. "MEASUREMET OF COUPLIG-COSTATS I DA FRAGMETS BY 2D MR.". J Magn Reson , Szyperski, T., Fernández, C., Ono, A., Kainosho, M. and Wüthrich, K. (1998) Measurement of Deoxyribose 3 J Scalar Couplings Reveals Protein-Binding Induced Changes in the Sugar Puckers of the DA. J. Am. Chem. Soc. 120, Iwahara J, Wojciak JM, Clubb RT. (2001), An efficient MR experiment for analyzing sugarpuckering in unlabeled DA: application to the 26-kDa dead ringer-da complex. J Magn Reson. 2001, 153, 262 Multinuclear experiments, DA/RA Pardi, A. and ikonowicz, E.P. (1992) Simple procedure for resonance assignment of t protons in 13C labeled RA J. Am. Chem. Soc., 114, Sklénar, V., Miyashiro,., Zon, G., Miles,.T., Bax, A. (1986) Assignment of the 31P resonances in oligonucleotides by two-dimensional MR spectroscopy FEBS Le 94 9 Varani, G., Aboul-ela, F., Allain, F., Gubser, C.C. (1995) ovel three-dimensional 1 1 triple resonance experiments for sequential backbone correlations in nucleic acid Biomol. MR, 5, Legault, P., Farmer, B.T. II, Mueller, L. and Pardi, A. (1994) Through-bond correlation adenine protons in a 13C-labeled ribozyme. J. Am. Chem. Soc., 116, Marino, J.P, Prestegard, J.. & Crothers, D.M. (1994) Correlation of adenine 2/8 re in uniformly 13C labeled RAs by 2d CC-TOCSY: a new tool for 1 assignm Chem. Soc., 116, Sklenár, V., Peterson, R.D., Rejante, M.R., Wang, E. & Feigon, J. (1993) Two-dimens resonance CC experiment for direct correlation of ribose 1 and Base 8, in 13C, 15-labeled RA oligonucleotides. J. Am. Chem. Soc., 115, Sklenár, V. Peterson, R.D., Rejante, M.R. & Feigon, J. (1994) Correlation of nucleotide sugar protons in 15 labeled IV RA oligonucleotide by 1-15 SQC experim Biomol. MR, 4, P Schmieder, J Ippel, van den Elst, G A van der Marel, J van Boom, C Altona, a Kessler (1992) eteronuclear MR of DA with the heteronucleus in natural abu facilitated assignment and extraction of coupling constants. ucleic Acids Res Schwalbe, J. P. Marino, G. C. King, R. Wechselberger, W. Bermel, and C. Griesinge "Determination of a complete set of coupling constants in 13C-labeled oligonucle Biomol. MR 4, Trantirek L., Stefl R., Masse J.E., Feigon J. and Sklenar V. (2002)"Determination of the torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling consta 3J(C2)/4-1' and 3J(C6)/8-1'" J. Biomol. MR., 23(1):1-12 Szyperski, T., Ono, A., Fernández, C., Iwai,., Tate, S., Wüthrich, K. and Kainosho, M Measurement of 3JC2'P Scalar Couplings in a 17 kda Protein Complex with 13 C Labeled DA Distinguishes the B I and BII Phosphate Conformations of the DA Chem. Soc. 119, Szyperski, T., Fernandez, C., Ono, A., Wüthrich, K. and Kainosho, M. (1999) The { 31P echo-difference Constant-time [ 13C,1 ]-MQC Experiment for Simultaneous Determination of 3 J3'P and 3JC4'P in ucleic Acids and their Protein Complex Magn. Reson. 140, Schwalbe, W. Samstag, J. W. Engels, W. Bermel, and C. Griesinger, (1993) "Determ 3J(C,P) and 3J(,P) Coupling Constants in ucleotide Oligomers", J. Biomol. M 486 C. Richter, B. Reif, K. Wörner, S. Quant, J. W. Engels, C. Griesinger, and. Schwalbe "ew Experiment for the Measurement of 3J(C,P) Coupling Constants including and 3J(C4'i,P i+1) coupling constants in Oligonucleotides" J. Biomol. MR 12, 22