Nucleic Acids NMR Spectroscopy. Markus W. Germann Departments of Chemistry and Biology Georgia State University
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1 2012 ucleic Acids MR Spectroscopy Markus W. Germann Departments of Chemistry and Biology Georgia State University
2 Sometimes a picture helps! o animals were hurt!!
3 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) B) C) D) E) 1 Chemical shifts! Assignment of exchangeable! Assignment of non-exchangeable proton! Typical Es in helical structures! Correlation between non-exchangeable!!and exchangeable protons!
4 MR Spectroscopy is an Important Method for Structural Studies of ucleic Acids: PDB olding, March 2, 2010! Technique! Molecule! Proteins! ucleic Acids! Protein/ucleic! Acid Complexes! ther! X-ray Diffraction! ! 1 193! 2 372! 17! ! MR! 7 219! 894! 153! 7! 8 273! ther 1)! 322! 23! 78! 14! 436! total! ! 2 109! 2 603! 38! ! 1) EM, ybrid, other!
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8 Alternate Bases & Modifications (small selection):
9 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, and functions (2000) By Victor A. Bloomfield, Donald M. Crothers, Ignacio Tinoco
10 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 Enzymology (1995), 261, ucleic acids: structures, properties, and functions (2000) By Victor A. Bloomfield, Donald M. Crothers, Ignacio Tinoco
11 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
12 Chemical shift ranges in nucleic acids! (G, T, U)! 2 (G, C, A)! A-T! G-C! Loops, MM! 2, 8, 6! 1! 3! 4,5,5! 2,2! DA! A-U! G-C! Loops, MM! 2, 8, 6! 1! 2,3,4,5,5! RA! 15! 10! 5! 0!
13 1' 5-6! 2' (A,G) (T,C)! 2'' (A,G) (T,C)! 3' ! 4' ! 5' ! 5'' ! C1' 83-89! C2' 35-38! C3' 70-78! C4' 82-86! C5' 63-68! DA! RA! 1' 5-6! 2' ' ! 4' ! 5' ! 5'' ! C1' 87-94! C2' 70-78! C3' 70-78! C4' 82-86! C5' 63-68!
14 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!
15 Adenine!!!Guanine! 2!7.5-8!C2! !-!-!C2!156! 8! !C8! !8! !C8! ! 6!5-6/7-8!6!82-84!1! !1! ! -!-!-!2!5-6/8-9!2!72-76!!!C4! !!!C4! !!!C5! !!!C5! !!!C6! !!!C6!161!!!1! !!!1! !!!3! !!3!167!!7! !!7! !!!9! !!!9! ! Thymidine!!!Uridine!!!Cytidine!!!! 6! !C6! !6! !C6! !6! !C6! ! Me5! !Me5!15-20!5! !C5! !5! !C5!94-99! 3!13-14!3!156!3!13-14!3! !-!-!3!210! -!-!-!-!-!-!!-!!4!6.7-7/81-8.8!4!94-98!!!C2 154!!!!C2!154!!!C2!159!!!C4 169!!!!C4!169!!!C4! !!!C5!95-112!!!!C5! !!!C5!94-99!!!1!144!!!!1! !!!1! !
16 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
17 DA airpin AT! GC! T! Thermal lability! ermann et al., ucl. Acids Res :
18
19 ew DA constructs Do the duplexes form, is there base pairing? Does the unusual base pair form? α!!!!!!! C1! C! 3! alphat2! 5 -G C G A A T α!t! C G C! C G C α!t! T A A G C G-5! alphag! 5 -G C α!g! A A T T C G C! C G C T T A A α!g! C G-5! 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 C G G C A A T T α!t! α!t! A A C G G C! 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!
20 T7 imino 1 MR G9 G3 T6 alpha G G1 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-5! ppm ppm
21 d-gcgaattcgc! CGCTTAAGCG-d! A! d-gcg! A! CGC! T! T! d-gcgaattcgc! CGCTTAAGCG-d! A! d-gcg! A! CGC! T! 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!
22 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
23 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: Easy to set up Excellent water suppression Cons: Resonances under water signal! (T variation) Labile protons not visible (some GC pairs may be) M y pulse to dephase the water Bsignal. z M =0 Selective rf pulse on solvent resonance followed by a gradient This could be followed by a mild presaturation field. The selective rf pulse (1-2ms, depending on width to be zeroed) is usually of the gauss type. The selective rf pulse z-gradient constructs could be repeated (WET).
24 Jump and return Watergate d1 90 x 90 -x td 90 x 180 x z y z y y 90 -x 90 -x Δ Δ Δ Δ x x G z p 1 G 1 + p 2 G 2... = 0 Water! Pros: Easy to set up Excellent water suppression (with proper setup as good as presat) Good for broad signals! Cons: on uniform excitation Baseline not flat Pros: Excellent water suppression Uniform excitation Baseline flat Cons: May loose broad resonances ther sequences: 1331 etc
25 Exitation Sculpting T.-L. wang & A.J. Shaka, J. Mag. Res. (1995), ! 1D! ESY! Pros: Easy to set up Excellent water suppression Good for broad signals! Uniform excitation Cons: May loose some intensity on very broad signals pectra: 1.5mM DA in Water, anjunda, Wilson and Germann unpublished!
26 Structure Determination, MR experiments: I) Assignment ESY, CSY, SQC 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 (E, CSY) inter strand/cross strand (E, CSY) dipolar coupling (SQC, SQC) Black: unlabeled, Blue: labeled DA or RA
27 Stereospecific Assignment!! 5!'!! 5!''!!! 3!'!! 2!'! B!! 4!'!! 1!'!!! 2!''!!! 5!'!! 5!''!!! 3!'!! 2!'!! 4!'! Deoxyribose!!! Ribose! B!! 1!'! ow do we determine them? 2! a) Rule of Thumb (5 downfield of 5 ) Shugar and Remin BBRC (1972), 48, ) b) Short mixing times ESY d1 2 shorter than 1 2 -> Crosspeak 1-2 > 1 2 2!
28 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
29 Distance information determines the glycosidic torsion angle 2.5Å! 3.8Å! ow do we get distance information? o uclear verhauser effect (< 6Å)
30 Distance information determines the glycosidic torsion angle 2.5Å! 3.8Å! ow do we get distance information? o uclear verhauser effect (< 6Å)
31 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: = β α δ γ ε 4 ν4 ν0 ν3 ν1 ν2 χ 3 endo! 2 endo!
32 (orthern)! 5! 3 endo! (Angle ~ 90 deg)! 5! 2 endo! S! (Angle ~ 170 deg (Southern)!
33 2 endo sugar 1, 2, 2, 3 region! 1! 3! 2! 2! Widmer,. and Wüthrich,K. (1987) J. Magn. Reson., 74,
34 3 endo sugar 1, 2, 2, 3 region! 1! 3! 2! 2!
35 CSY! 2! 1! 1! 3! 2 endo sugar! 2 endo sugar 1, 2, 2, 3 region! 2! 2! 1-2! 1-2! 2! 3 endo sugar! 2! 1-2! 1-2! 2!
36 LFA- CSY! 2 endo sugar 1, 2, 2 region!
37 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 = (Σ 1 9.8)/5.9 See also our pure examples: f S =0 and ~1 respectively Σ 1 = J J 1 2 Σ 2 = J J J 2 2 Σ 2 = J J J 2 2 Σ 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
38 Sugar puckering control alphat t Σ1 fs Σ1 fs C G C T α!t! alphat! α!t! A A G C G-5! G C G A A T T (15.3) (0.93) C G C10 (14) (0.7) (14) (0.7) (14) (0.7) (14) (0.7) (14) (0.7) ramini, 2000, J. Biomolecular MR, 18, MD calculation! MD-Tar calculation!
39 Pseurot calculations! Φ S,!= 37! P S!= f S!= Φ S,!= 37! P S!= ! f S!= ramini, et al., 1998, ucleic Acid Research, 26, van Wijk,J., aasnoot,k., de Leeuw,F., uckreide,d. and Altona,C. (1995) PSEURT 6.2. A Program for the Conformational Analysis of Five Membered Rings. University of Leiden, The etherlands
40 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
41 Sugar Puckering from Cross-Correlated Relaxation Γ DD-DD Γ C1 1 -C2 2 = k (3cos2 θ-1)τ c θ = 180: for 2 endo (B form) Large and positive 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 form) Small and negative
42 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 DA. 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 Biomacromolecules Chapter 7 p Christian Griesinger (also for α and ζ)
43 nucleotide unit 3 β α γ δ ε ν4 4 ν3 ν0 ν1 ν2 χ α and ζ pose problems! Determinants of 31 P chem shift.! ε and ζ correlate. ζ = ε! ζ 5
44 Backbone Experiments: CT-ESY, CT-CSY Bax, A., Tjandra,., Zhengrong, W., ( 2001). Measurements of 1-31 P dipolar couplings in a DA oligonucleotide by constant time ESY difference spectroscopy, J. Mol. Biol., 19,
Nucleic Acids NMR Spectroscopy. Markus W. Germann Departments of Chemistry and Biology Georgia State University
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