Zjišťování struktury DNA pomocí NMR a molekulových simulací Jiří Kozelka kozelka.jiri@gmail.com 4.10.2013 Použitý materiál: http://chemistrv.osu.edu/~foster.281/biochern766/download/PDF f i les/7 66 nmrlecture.ppt.pdf http://www.chem.ufl.edu/~nmr/lon/2D-1 .pdf http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/COURSE_2012/ Lecture_NucleicAcids_2012.pdf Doporučené speciální kurzy: C8950 NMR - Strukturní analýza, prof. RNDr. Radek Marek, Ph.D. Doporučená literatura: Wüthrich, K, 1984 "NMR of Proteins and Nucleic Acids" J. Wiley & Sons, NY. NMR Spektroskopie I 1 o Využívá jaderného spinu nuklidů (]H, 13C,31P,15N) I Ve stacionárním magnetickém poli B0 preferují spiny energeticky výhodnou polohu paralelní s B0 Dodáním potřebné energie AE přejde spin do excitovaného stavu (poloha antipařalelní s Bo): AE=hyB0/27i h: Planckova konstanta, : magnetogyrický faktor (charakteristický pro daný nuklid) AE dodáme v podobě elektromagnetického záření o frekvenci v: hv= hyB0/27i v= yB0/27r V molekule je magnetické pole B0 modulováno magnetickými momenty elektronů: B0^ BL (lokální)=(l-CT) B0 a: „stínící konstanta", charakteristizuje okolí daného jádra frekvence, při které nastává rezonance: vL= yBL/27C vL je charakteristická při daném B0 pro jádro v molekule Jako standard používáme pro XH NMR např.: CH3 CH3 I I H3C—Si—CH3 Na+ 03S—CH2-CH2-CH2—Si—CH3 Tetrametylsilan (TMS) rozpustný v org. rozpouštědlech 4,4-Dimeťyl-4-silapentansulfonan sodný (DSS) rozpustný ve vodě Rezonanční frekvenci udáváme zpravidla „chemickým posunem" 5: ô=lO6. (VL-vťef)/vťef [ppm] = 106. (BL-Bref)/Bief (vreí=vTMS nebo vDSS) Bl=(1-ctl) B0 Brer(l-CTťef) B0 (l-^)^o 1-ťT. -l06^(aref-aL)-\06[ppm] ref Faktory ovlivňující 5: 1* Indukční efekt elektronegativních atomů 5(1H) [ppm] CH3J 2.16 CH3Br 2.68 CH3C1 3.05 CH3F 4.26 2* Magnetická anisotropic dvojných vazeb Aldehydic protons deshielded by a combination of inductive and anisotropy effects 6 » 10 ppm In the molecular plane: Induced field of jr-electrons parallel to B0: deshielding Below and above the molecular plane: Induced field of 71-electrons antiparallel to B0: shielding Faktory ovlivňující 6 (pokračování): 3. Kruhové proudy aromatických jader Region 4. Polarizační efekt nabitých atomů stínění nebo odstínění vždy odstínění podle polohy nabité skupiny (deshieldng, A8>0) (ve znázorněném případě odstínění) 1D 1H NMR spectrum of DNA The assignment problem: Which peak corresponds to which atom in the molecule? CH, (ppm) Figure 5-5 The one-dimensional ID specirum of the DNA oligomer d(CGCAAAAGGC) • d(GCCTTTTGC*G) is shown (20 C in H,0 solution, solvent suppressed by prcsaturation>. with the regions of the spectrum labeled. The imino and amino resonances of the terminal base pairs are missing due to exchange with solvent. Peaks marked with an x arc small molecule contaminants. 2D NMR Spectroscopy allows magnetic interactions (correlations) between spins to be detected and depicted in a 2-dimensional diagram. 2 D spectra are 3D objects Cross-peaks reveal a correlation between the frequencies on the two frequency axes. 1-2 1.6^ 1.7 II 11 I I I I | I I I 11 I I 11 II I | I I II | I II 11 I I 11 II I | I I I I | I I 11 II I 11 II I | I I II | I I II | II I 11 II I I I I I | I II Correlations: - Through bonds - based on scalar coupling. - Through space - based on dipolar relaxation (nOe's). NOE = Nuclear Overhauser Effect ISC 160 HO 120 100 30 60 40 20 F1 (mm) Sequential Resonance Assignments in lH NMR Spectra of Oligonucleotides by Two-Dimensional NMR Spectroscopy"* 265 citations by 2.10.2013 R. M. Scheek, R. Boelens, N. Russo,* J. H. van Boom,* and R. Kaptein* 2D NOESY spectrum of d(TGAGCGG)-d(CCGCTCA) three 2-D NOE spectra with mixing times rm = 80, 120, and 160 ms (serves as guideline for distances, this that were recorded to obtain NOE buildup curves as shown in Figure is not a fragment of the studied DNA 9. duplex) Sequential Resonance Assignments in *H NMR Spectra of Oligonucleotides by Two-Dimensional NMR Spectroscopy"* R. M. Scheek, R. Boelens, N. Russo,* J. H. van Boom,8 and R. Kaptein* 2D COSY spectrum of d(TGAGCGG)-d(CCGCTCA) H27H2" H47 H57H5" H3'u H5/H1' H6/H86. H6/H8 5 5 H47 * ! H5/H1'ppwH3' H57H5« H27H2" figure 3: 500-MHz COSY spectrum of the 7 bp DNA duplex. A total of 64 scans was recorded for each FID, The boxed regions correspond to those in Figure 1. Canonical 5'-GT-3' fragment of B-DNA (serves as guideline for distances, this is not a fragment of the studied DNA duplex) Chemical shifts of the non-exchangeable protons in a double-stranded DNA heptamer 5'-TGAGCCG-3' 3'-ACTCGGC-5' BIOCHEMISTRY scheek et Tabic II: Chemical Shifts of Assigned lH Resonances (in ppm, Relative to DSS) in the Duplex of d(TGAGCGG) and d(CCGCTCA) H8 or H6 CH5 or TCH3 HI' H2' H2" H3' H4' H5',H5"° Tl 7.31 1.58 5.74 1.73 2.18 4.62 4.02 NA G2 7.99 5.36 2-74 2-80 4.98 4.31 4.04, 3.95 A3 8.18 6.06 2.74 2.90 5.08 4.46 4.22,4.14 G4 7.69 5.75 2.50 2.62 4.98 4.39 4.21,4.20 C5 7.25 5.28 5.67 1.79 2.25 4.79 4.12 NA G6 7.82 5.65 2.62 2.71 4.95 4.33 4.07, 3.98 G7 7.73 6.10 «rc WWrJ 4.65 4.22 NA CI 7.65 5.78 5.86 2.02 ■ ■ 2.47 4.63 4.10 NA C2 7.52 5.64 5.58 2.12 2.43 4.86 4.15 NA G3 . 7.94 5.95 2.71 2.76 5.02 4.41 4.14,4.06 C4 7.44 5.40 5.96 2.08 2.52 4.74 4.25 NA T5 7.43 1.65 6-05 2.08 2.42 4.86 4.16 NA C6 7.50 5.78 5.70 2-03 2.25 4.82 4.09 NA A7 8.24 6.30 2.16 "2.48' 4.71 4.21 NA NA, not assigned. Scheek, R. M.; Boelens, R.; Russo, N.; van Boom, J. H.; Kaptein, R. Biochemistry 1984, 23, 1371 -1376. to desh 3'-terminus: No 3'-phosphate to deshield H2" Model of B-DNA (4 base-pairs) I - —« H2" deshielded by 3'-phosphate Chemical shifts of the non-exchangeable protons in a double-stranded DNA heptamer 5'-TGAGCCG-3' 3'-ACTCGGC-5' 1374 BIOCHEMISTRY scheek et Tabic II: Chemical Shifts of Assigned 'H Resonances (in ppm, Relative to DSS) in the Duplex of d(TGAGCGG) and d(CCGCTCA) H8 or H6 CH5 or TCH3 HI' H2' H2" H3' H4' H5',H5"a Tl 7.31 1.58 5.74 1.73 2.18 4.62 4.02 NA G2 7.99 5.36 2.74 2.80 4.98 4.31 4.04, 3.95 A3 8.18 6.06 2.74 2.90 5.08 4.46 4.22,4.14 G4 7.69 5.75 2.50 2.62 4.98 4.39 4.21,4.20 C5 7.25 5.28 5.67 1.79 2.25 4.79 4.12 NA G6 7.82 5.65 2-62 2.71 4.95 4.33 4.07, 3.98 G7 7.73 6.10 «W ►*W 4.65 4.22 NA CI 7.65 5.78 5.86 2.02 2.47 \ 4.63 4.10 NA C2 7.52 5.64 5.58 2.12 2.43 \ 4.86 4.15 NA G3 . 7.94 5.95 2.71 2.76 \ 5.02 4.41 4.14,4.06 C4 7.44 5.40 5.96 2.08 2.52 \ 4.74 4.25 NA T5 7.43 1.65 6.05 2.08 2.42 \ 4.86 4.16 NA C6 7.50 5.78 5.70 .2.03, ,2-25 \ 4.82 4.09 NA A7 8.24 6.30 T70" T48 \ 4.71 4.21 NA a NA, not assigned. Scheek, R. M.; Boelens, R.; Russo, N. Biochemistry 1984, 23, 1371-1376. van Boom, J. H.; Kaptein, R. 3 -terminal nucleotide: no deshielding of H2" by the 3'-phosphate Assignments of non-exchangeable protons: Sekvenční konektivity 5'—>3'cukr-báze kroku 5 -GpT-3' v kontextu B-DNA 5-G 3-T Konektivity 3—»5 jsou podstatně delší! d(GCGAATTaCGC)2 d(GCGAATTaCGC)2 Strukturní informace získaná ze spekter NMR 1. Odstupy mezi atomy ze spekter NOESY (Nuclear Overhauser Effect Spectroscopy): intenzita cross-peaku x r6 2. Dihedrální úhly odvozené ze spin-spinových konstant získaných ze spekter COSY (Correlated Spectroscopy) pomocí Karplusovy rovnice 3. Informace získané z chemických posunů 1., 2: Je možno zohlednit ve výpočtech molekulového modelování jako „restraints" 3.: Kvantifikace vlivu okolí na chemický posun je komplikovaná, proto jej těžko lze zohlednit automaticky při výpočtu struktury. Velikost vicinálních spin-spin-interakčních konstant závisí na dihedrálním úhlu i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i 0 20 40 60 80 100 120 140 160 180 V jiných pramenech má Karplusova rovnice tvar uvedený dole. Ukažte, že obě rovnice jsou ekvivalentní a vypočtěte vztah mezi koeficienty A, B, C a P, Q, R 3JHH = Pcos2<|> + Qcosc|> + R http://vvww2.chemistry.msu.edu/faculty/reusch/virttxtjml/spect Strukturní informace získaná ze spekter NMR 1. Odstupy mezi atomy ze spekter NOESY (Nuclear Overhauser Effect Spectroscopy): intenzita cross-peaku x r6 2. Dihedrální úhly odvozené ze spin-spinových konstant získaných ze spekter COSY (Correlated Spectroscopy) pomocí Karplusovy rovnice 3. Informace získané z chemických posunů 1., 2: Je možno zohlednit ve výpočtech molekulového modelování jako „restraints" 3.: Kvantifikace vlivu okolí na chemický posun je komplikovaná, proto jej těžko lze zohlednit automaticky při výpočtu struktury. Molecular modeling is an ensemble of methods for the study of molecular conformations, based on energy calculations. It comprises geometry optimizations by minimizing the energy and molecular dynamics simulations. Empirical energy calculation: bonds angles v. + -y(l+cos(nco-y)) torsions N N i=l j=i+l 1j 12 a y y y y + j 47ra0ry Two main techniques are used to optimize the geometry of a molecular model: 1. Molecular mechanics calculations: Using an optimization algorithm, we move the atomic coordinates so as to minimize the empirical potential. 2. Molecular dynamics simulations: We assign random velocities to the atoms, whose mean square corresponds to a chosen temperature. Then, in infinitesimal steps, we let the atoms move in the empirical potential. MD simulations allow us: - to describe the dynamical behaviour of the molecule and to include solvent - to overcome energy barriers - to estimate the Gibbs free energy of the solvated molecule bonds angles v. + -y(l+cos(nco-y)) torsions N N i=l j=i+l 1j 12 a y y y y + j 47ra0ry Principal problem of Molecular mechanics calculations: For most of the molecules, many conformations close in energy exist, and our calculations are not accurate enought to define which conformation corresponds to the global minimum. Therefore, we have to check the calculated model against experimental structural data. This can be done by adding to the empirical energy a „penalty function", accounting for discrepancies between experiment and the calculated model. bonds angles v. + -y(l+cos(nco-y)) torsions N N i=l j=i+l 1j 12 a y y y y + j 47ra0ry From NOESY spectra, we usually obtain upper and lower limits for a given interatomic distance dj5 djuPPerand djlower. A pseudo-energy term is then added for distances higner than the upper limit or lower than the lower limit: rpupper ^distance E upper AAdj — d^pper)2 if cL >ct; pper 0 3 " -J otherwise. jplower distance E I o tower I - d'™^)2 if dj- < dl°wer otherwise. Similar penalty terms are added for deviations of dihedral angles from the experimental upper and lower limits. Structural Parameter: The Sugar Pucker 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 (T). The geometry is defined by two parameters: the pseudorotation phase angle (P) and the pucker amplitude (O). The pseudorotation cycle of the furanose ring shows the relationships among the phase angle of pseudoration (P) and the alternative E/T (envelope/twist) and emfaiexo notations. Conformations corresponding to the northern half are designated as N-type and those corresponding to the southern half are designated as S-type. The P values commonly observed for the N and S conformations are represented by CV-endo (3E) and C2'-endo (2E), respectively. 32 Measuring the Sugar Pucker by NMR Very weak JH1._H2' and strong JH3-.H4i cross-peaks correspond to pure N-type conformation (preferred conformation in RNA). Strong JHi'-H2' and weak JH3< H4- cross-peaks correspond to pure S-type conformation. Jh2'-h3' is similar in both states. Intermediate intensities indicate an equilibrium between N and S states. P-DS-1E* Ribose:3J HJ-Hi I Y-.z Figure 27. Karpius relation of 'MHI'tfZ), Jl(H2',HIl and Ji(HJ,H4) coupling constants depending on the pseudafotalion phase P at apseudorotation amplitude of 44". J 1 14 .1 ID Ha Penac sugar H1\ H2". H2H region 5 . 6 B.I - J Hi1-Hi" I } ill » J B1-H2' 2.« F VI i^S ifli i.Bl S.U 5«1 SJfl i7S iTI 5.JT S.7t ffB Structural Parameter: The Glycosidic Torsion Angle Measuring Using Distances The base can exist in 2 distinct orientations about the N-glycosidic bond. These conformations are identified as. syn and and. The attti conformation predominates. NH, H f' N -H HO OH Aitri Adenosine HOCH, s . HO OH Syn-Adenosine Distance information (H8 - HI') determines the glycosidic torsion angle: anii Furthermore, although H8 resonances from nucleotides in the anil glycosidic conformation are in close contact to several sugar proton resonances, in the syn conformation no close contact to sugar protons is expected (except to the anomeric resonances). 34 Measuring the Glvcosidic Torsion Angle Using Scalar Couplings Base-to-sugar three-bond C6/C8-HF and CV- H6/H8 coupling constants can also be used to define the glycosidic angle %: i ii SYfl .'hi row anti -x- syti ISO 270 36Q W-C2 WC,T) 'JH1'-C8 (A,G) >JH1'-C4 (A,G) Příklad využití technik NMR pro strukturní studii Unexpected intrastrand-to-interstrand rearrangement of Pt-GG crosslinks formed between an analogue of the antitumor drug cisplatin and a DNA duplex: Evidence for kinetic instability of Pt-N bonds Kubicek, K.; Monnet, J.; Scintilla, S.; Kopecna, J.; Arnesano, R; Trantirek, L; Chopard, C; Natile, G.; Kozelka, J. Chem. Asian J. 2010, 5, 244-247 Context Cisplatin and Oxaliplatin are efficient antitumor drugs with different cytotoxic properties.1 The differences in their biological effects are believed to be related to different structural perturbations they cause to their cellular target, DNA. Oxaliplatin contains a chiral diamine ligand, and the question arises how the chirality affects the adduct structure. Cl HjN—Pt-Cl NH> H2N NH, 9fl R.R-TJ mm Ä,Ä-DAC'H We are therefore investigating DNA oligonucleotides bearing adducts of c/s-Pt(NH3)22+, Pt(fl,fl-DACH)2+, and Pt(S,S-DACH)2+, using NMR spectroscopy and molecular modeling. /\ CCTT TCTC G GA A C C A G A G HjN HjN A CCTT TCTC GGAACCAGAG V CCTT TCTC GGAACCAGAG 1 E. Raymond, S. Faivre, S. Chaney, J. Woynarowski, E. Cvitkovic, Mol.Cancer Ther. 2002, 1, 227-235. An unexpected result In this work, we have prepared and purified the adduct with Pt(S,S-DACH)2+. During the NMR analysis, we observed that the intrastrand crosslink reversibly rearranged to a new species that we identified as an interstrand crosslink with the 5'-terminal G. CCTT TCTC CCTTG TCTC/ GGAAC CAGAG GGAACCAGA A simple method to investigate the interstrand crosslink separately: heating to 40 °C At room temperature, the intrastrand and interstrand crosslinks form an approximately 1:1 equilibrium. Above 15 °C, the intrastrand crosslink starts to melt and at 40 °C, its signal coalesce and disappear from the NOESY spectra. At this temperature, it was thus possible to study the interstrand crosslink separately Peaks of newly formed species _ (turned out to be a DNA duplex containing an interstrand crosslink). They correspond to a strongly shielded proton with many contacts. 500 ms NOESY spectra of a -2.6 mM solution pH 7.2 (20 mM phosphate) containing 1 and 2 E 4- CL Q. peaks of initial species (Pt-crosslinked DNA duplex) 8 8 0 9 I i 0 i'I V 1 Cíl .» i I I 0 * 8 o o 0 ic ■2 0 t i » • i 0 v < i i i H ídditi} « 0 0 O -4 cfb¥° o 'o ' a -6 8 500 ms NOESY spectra of a -2.6 mM solution pH 7.2 (20 mM phosphate) containing 1 and 2 Interstrand crosslink 3D model of the interstrand crosslink shows a very unusual DNA structure The methyl group of T7 (green) is intercalated between the two platinated guanines (yellow), with many contacts to other protons (blue). This intercalation resembles to that seen in recognition complexes between cellular proteins and kinked DNA. The intercalation of thymine CH3 between two guanines explains the strong shielding of this methyl group! Conclusion Pt-N bonds are kinetically unstable and can break at physiologically relevant rate. Kubicek, K.; Monnet, J.; Scintilla, S.; Kopečná, J.; Arnesano, R; Trantirek, L; Chopard, C; Natile, G.; Kozelka, J. Chem. Asian J. 2010, 5, 244-247