Nucleic Acids NMR Spectroscopy. Markus W. Germann Departments of Chemistry and Biology Georgia State University

Transcription

1 ucleic Acids MR Spectroscopy Markus W. Germann Departments of Chemistry and Biology Georgia State University

2 MR of ucleic Acids 1 1) MR Spectroscopy for Structural Studies 2) Primary Structure of DA and RA 3) Resonance Assignment of DA/RA by omonuclear MR A) 1 Chemical shifts B) Assignment of exchangeable C) Assignment of non-exchangeable proton D) Typical Es in helical structures E) Correlation between non-exchangeable and exchangeable protons

3 MR Spectroscopy is an Important Method for Structural Studies o ucleic Acids: PDB olding, March 2, 2010 Technique Molecule Proteins ucleic Acids Protein/ucleic Acid Complexes ther X-ray Diffraction MR ther 1) total ) EM, ybrid, other

4

5 Sugar Sugar Sugar umbering

6 Sugar Sugar umbering

7 Alternate Bases & Modifications (small selection):

8 ETER BASE PAIRS 3 C + oogsteen 3 C Watson-Crick 3 C Reverse Watson-Crick Germann et al., Methods in Enzymology (1995), 261, ucleic acids: structures, properties, an functions (2000) By Victor A. Bloomfiel Donald M. Crothers, Ignacio Tinoco

9 3 C C 3 C 3 C 3 M BASE PAIRS + CC + TT(I) TT(II) AA(I) GG(I) AA(II) GG(II) 2 2 Germann et al., Methods in Enzymolog (1995), 261, ucleic acids: structures, properties, an functions (2000) By Victor A. Bloomfie Donald M. Crothers, Ignacio Tinoco

10 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.. E accuracy > account for spin diffusion Backbone may be difficult to fully characterize > especially α and ζ. Dipolar couplings

11 Chemical shift ranges in nucleic acids (G, T, U) 2 (G, C, A) A-T G-C Loops, MM 2, 8, ,5,5 2,2 DA A-U G-C Loops, MM 2, 8, 6 1 2,3,4,5,5 RA

12 1' 5-6 2' (A,G) (T,C) 2'' (A,G) (T,C) 3' ' ' '' C1' C2' C3' C4' C5' DA RA 1' 5-6 2' ' ' ' '' C1' C2' C3' C4' C5' 63-68

13 9R-Borano DA RA 5 -d(a T G G T G C T C) (u a c c a c g a g)r-5

14 Adenine Guanine C C C C / / C C C C C C Thymidine Uridine Cytidine C C C Me Me C C / C2 154 C2 154 C2 159 C4 169 C4 169 C C C C

15 o Structure Required! ften, depending on the question asked, a full structure determination is not required! Does it form a duplex?! Which base pairs are thermo labile?! Which base pair is which assignment?! Is the loop structured?! Structure

16 DA airpin AT GC T Thermal lability ermann et al., ucl. Acids Res :

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18 ew DA constructs! Do the duplexes form, is there base pairing?! Does the unusual base pair form? 5 -G C G A A T α T C G C alphat2 C G C α T T A A G C G-5 α C1 alphag 5 -G C α G A A T T C G C C G C T T A A αg C G-5 C 3 alphaa 5 -G C G α A A T T C G C C G C T T A α A G C G-5 C 3 alphac 5 -G C G A A T T α C G C C G α C T T A A G C G-5 β C1 ramini & Germann, Biochem cell biol. 1998, 76, alphat control 5 -G C G A A T α T C G C C G C α T T A A G C G-5 5 -G C G A A T T C G C C G C T T A A G C G-5

19 T7 imino 1 MR G9 G3 T6 G1 alpha G T6 T7 G9 G1 G3 alpha A T7 T6 G9/G1 G3 alpha C T6 T7 G1 G9 G3 alpha T T7T6 G9/G1 G3 control 5 -G C G A A T T C G C C G C T T A A G C G ppm

20 Local Parallel Stranded Environment is ecessary for Stable Duplex Formation d-gcgaattcgc CGCTTAAGCG-d d-gcg CGC A A T T d-gcgaattcgc CGCTTAAGCG-d A d-gcg CGC T B A: dt 11 B: d(ccgg) 2 C: alphat (0.5 µm) D: control (0.5 µm) E: alphat (115 µm) F: control (115 µm) A d-gcg A CGC T T A BPB 15% ative PAGE, 15 mm MgCl 2

21 Solvent Suppression The presence of an intense solvent resonance necessitates an impractical high dynamic range. 110 M vs <1mM (down to 5-10 um) To overcome this problem several methods are currently applied: 1) Presaturation 2) bserving the FID when the water passes a null condition after a 180 degree pulse. 3) Suppression of broad lined based on their T 2 behavior. 4) Selectively excitation, with and without gradients 5a) Use of GRASP to select specific coherences thereby excluding the intense solvent signal. In this case the solvent signal never reaches the ADC. This allows the observation of resonances that are buried under the solvent peak. 5b) Use of GRASP to selectively dephase the solvent resonance (WATERGATE) 5c) Excitation sculpting

22 Presat P18 90 Selective Excitation 90 P18 x td 180 y td SIGLE LIE RESACE Presaturation field strength: z corresponds to a 6-12ms 90deg pulse B o B z Pros: Cons: Easy to set up Excellent water suppression Resonances under water signal! (T variation) Labile protons not visible (some GC pairs may be) M y M =0 Selective rf pulse on solvent resonance followed by a grad pulse to dephase the water Bsignal. z This could be followed by a mild presaturation field. selective rf pulse (1-2ms, depending on width to be zeroed usually of the gauss type. The selective rf pulse z-gradient constructs could be repe (WET).

23 Jump and return Watergate d1 90x 90-x td 90 x 180 x z y z y y 90-x 90-x Δ Δ Δ Δ x x solvent G z p 1 G 1 + p 2 G 2... = 0 Water Pros: Cons: Easy to set up Excellent water suppression (with proper setup as good as presat) Good for broad signals! on uniform excitation Baseline not flat Pros: Cons: Excellent water suppression Uniform excitation Baseline flat May loose broad resonances ther sequences: 1331 etc

24 Exitation Sculpting T.-L. wang & A.J. Shaka, J. Mag. Res. (1995), D ESY Pros: Cons: Easy to set up Excellent water suppression Good for broad signals! Uniform excitation May loose some intensity on very broad signals pectra: 1.5mM DA in Water, anjunda, Wilson and Germann unpublished

25 Structure Determination, MR experiments: I) Assignment ESY, CSY, SQ TCSY II) Local Analysis glycosidic torsion angle (E, CSY) sugar puckering (CSY, CSY, E, backbone conformation (CSY, +) base pairing (E, CSY) III) Global Analysis sequential inter strand/cross strand dipolar coupling (E, CSY) (E, CSY) (SQC, SQC) Black: unlabeled, Blue: labeled DA or RA

26 Stereospecific Assignment 5 ' 5 '' B 2 3 ' 2 ' 4 ' 2 '' 1 ' Deoxyribose 2 5 ' 5 '' 3 ' 2 ' B ow do we determine them? 4 ' 1 ' a) Rule of Thumb (5 downfield of 5 ) Shugar and Remin BBRC (1972), 48, ) Ribose b) Short mixing times ESY d1 2 shorter than 1 2 -> Crosspeak 1-2 > 1 2

27 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.. E accuracy > account for spin diffusion Backbone may be difficult to fully characterize > especially α and ζ. Dipolar couplings

28 Distance information determines the glycosidic torsion ang 2.5Å 3.8Å! ow do we get distance information? o uclear verhauser effect (< 6Å)

29 Distance information determines the glycosidic torsion ang 2.5Å 3.8Å! ow do we get distance information? o uclear verhauser effect (< 6Å)

30 Sugar puckering The five membered furanose ring is not planar. It can be puckered in an envelope form (E) with 4 atoms in a plane or it can be in a twist form. The geometry is defined by two parameters: the pseudorotation phase angle (P) and the pucker amplitude ( ). In general: RA (A type double helix) C3' endo. DA (B type double helix) C2' endo. j = m cos (P (j-2)) m range: = "! # $ % 4 (4 (0 (3 (1 (2 ' 3 endo 2 endo

31 (orthern) 5 3 endo (Angle ~ endo S (Angle ~ (Southern)

32 2 endo sugar 1, 2, 2, 3 region Widmer,. and Wüthrich,K. (1987) J. Magn. R 74,

33 3 endo sugar 1, 2, 2, 3 region

34 CSY endo sugar 2 endo sugar 1, 2, 2, 3 region endo sugar

35 LFA- CSY 2 endo sugar 1, 2, 2 region

36 Sugar puckering Usually (DA) one observes equilibrium of the S and forms sugar repuckering. Unless one form greatly dominates the local analysis requires quite a few parameters: P, PS,, S, fs Several methods for analysis exist, graphical and the more rigorous simulation. In practice the desired outcome determines the effort to be made. Sums of the coupling constants are often easier to obtain. f S = (! 1 9.8)/5.9 See also our pure examples: f S =0 and ~1 respectively! 1 = J J 1 2! 2 = J J J 2 2! 2 = J J J 2 2! 3 = J J J 3 4 If fs < 50% J 1 2 < J 1 2 If fs ca 0% J 1 2 very small If fs > 70% J 1 2 > J 1 2

37 Sugar puckering control alphat t!1 fs!1 fs G C G A A T T C G C10 (14) (0.7) (14) (0.7) alphat 5 -G C G A A T α T C G C C G C α T T A A G C G-5 ramini, 2000, J. Biomolecular MR, 18, MD calculation MD-Tar calculation

38 Pseurot calculations Φ S, = 37 P S = 125 f S = 0.4 Φ S, = 37 P S = 130 f S = 0.7 ramini, et al., 1998, ucleic Acid Research, 26, van Wijk,J., aasnoot,k., de Leeuw,F uckreide,d. and Altona,C. (1995) P A Program for the Conformational An Five Membered Rings. University of etherlands

39 Introduction to Cross-Correlated Relaxation Relaxation in MR determines experimental strategies and experiments dynamic and structural parameters Mechanisms Dipole -dipole CSA (e.g. 31 P at higher fields; proportional to B 2 ) Scalar relaxation (first and second kind) paramagnetic, etc Recently it became possible to use cross correlated relaxation (CCR) to directly measure bond angles without using a calibration curve as is needed for J s. DD -DD DD -CSA θ

40 Sugar Puckering from Cross-Correlated Relaxation Γ DD-DD Γ C1 1 -C2 2 = k (3cos 2 θ-1)τ c θ= 180: for 2 endo (B fo Large and posit Pseudorotation Phase Angle θ 1 2 = ψ m cos(p-144 ) iomr in Drug Research (2003) Chapter 7 p Christian Griesinger θ= 90: for 3 endo (A fo Small and negat

41 Sugar puckering: Summary -Coupling constants: CSY, E.CSY, low flip angle CSY omonuclear, eteronuclear -CT ESY -CSA-DD and DD-DD cross correlated data - 13 C chemical shifts, in favorable cases Some references Szyperski, T., et al. (1998). JACS. 120, Measurement of Deoxyribose 3 J Scalar Couplings Reveals Protein-Binding Induced Changes in the Sugar Puckers of the D Iwahara J, et al. (2001), J.Mag Res. 2001, 153, 262 An efficient MR experiment for analyzing sugar-puckering in unlabeled DA:. Couplings via constant time ESY. J. Boisbouvier, B. Brutscher, A. Pardi, D. Marion, and J.-P. Simorre (200), J. Am. Chem. Soc. 122, MR determination of sugar-puckers in nucleic acids form CSA-dipolar cross correlated relaxation, BioMR in Drug Research 2003 Editor(s): liver Zerbe (Wiley-VC) Methods for the Measurement of Angle Restraints from Scalar, Dipolar Couplings and from Cross-Correlated Relaxation: Application to Biomacromo Chapter 7 p Christian Griesinger (also for α and ζ) Summary to get J s E. Cosy DQ/ZQ principle FIDS Principle Quant. J correlation

42 nucleotide unit 3 "! $ # % (4 4 (3 (0 (1 (2 ' α and ζ pose problems Determinants of 31 P chem shift. ε and ζ correlate. ζ = ε & 5

43 Backbone Experiments: CT-ESY, CT-CSY Bax, A., Tjandra,., Zhengrong, W., ( 2001). Measurements of 1-31P dipolar couplings in a DA oligonucleotide by constant time ESY difference spectroscopy, J. Mol. Biol., 19,