Structural characterization of complexes using NOESY experiments

Transcription

1 Structural characterization of complexes using NOESY experiments Isotope labeling ( 3 C, 5 N) allows observation of individual components of a (binary) complex) H, 3 C, 5 N H, 3 C, 5 N 2 H/ H, 2 C, 4 N 2 H/ H, 2 C, 4 N Complex with different labeling of the components Isotope edited NMR spectra Isotope filtered NMR spectra

2 Basic terminology Isotope editing: selection of protons directly bound to a NMR active heteronucleus (e.g. 3 C- H or 5 N- H) Isotope filtering: suppression of protons directly bound to a NMR active heteronucleus, ideal suppression corresponds to the 'selection' of protons bound to NMR 'inactive' nuclei (e.g. 2 C- H, 4 N- H, O-H) Important isotopes for biomolecular NMR NMR active isotope natural abundance corresponding NMR inactive isotope natural abundance H % 3 C. % 5 N 0.37 % 2 H 0.05 % 2 C 98.9 % 4 N 99.63% ( 4 N, 2 H are quadrupol nuclei, relaxes very fast in biomacromolecules, nearly NMR inactive)

3 In practice: real isotope composition of complexes (in H 2 O solution) {95-99 % 5 N - H} {90-99 % 3 C- H} 00% O-H, S-H {-0% 2 C- H} { 99.7% 4 N - H} {99% 2 C- H} 00% O-H, S-H {% 3 C- H}

4 Selectivity for isotope editing Selectivity = Fraction of wanted observed component Fraction of unwanted observed component Sel. Iso.Ed. = concentration(labeled component) * degree of labeling concentration(unlabeled component) * (natural abundance) Sel. Iso.Filt. = concentration(unlabeled component) concentration(labeled component) * (fraction of protons not bound to a NMR active heteronucleus

5 Examples for the selectivity in a : complex Degree of 3 C labeling selektivity Iso.Ed. selektivity Iso.Filt. 50 % % % Isotope filtering requires a very high degree of isotopic labeling especially for NOESY experiments, because the relative intensity of different NOESY cross peaks show up factors in the range 0-00 (strong and weak NOE cross peaks!)

6 Basic principle for isotope filtering and editing H - X: H x J HX 2H z t H x cos( J HX t) + 2H y sin( J HX t) H : H x t H x Isotope editing: Isotope filtering: Selection of the term 2H y Suppression of the term 2H y and of the term H x for the HX pairs Additional requirements on filtering compared to editing

7 Basic principle for isotope editing H x J HX 2H z t H x cos( J HX t) + 2H y sin( J HX t) Maximizing of the 2H y term by setting t = / (2 J HX ) A: ± 90 H x H x cos( J HX t) + 2H y sin( J HX t) H x cos( J HX t) ± 2H z sin( J HX t) Alternating of receiver phase eliminates H x term B: H x cos( J HX t) ± 2H z sin( J HX t) ± 90 X y H x cos( J HX t) ± 2H z X x sin( J HX t) Alternating of receiver phase eliminates H x term At least one of both phase alternations is found in every phase cycling of an isotope editited experiment (e.g. HSQC)!

8 Dependence on the relative size of the J-coupling H x J HX 2H z t H x cos( J HX t) + 2H y sin( J HX t) J 0 = 40 Hz t = 3.57 ms J / J0 J / J0 y = cos( J HX t) y = sin( J HX t) Intensity of the H x -term is more sensitive to variations of the J couplings that means: Isotope filtering is more difficult compared to editing

9 Examples for filtering and editing H, I t / 2 t / 2 H z H y x ±x I z I y cos( J IS t) ± 2I x S z sin( J IS t) S Receiver + / + Filtering Receiver + / - Editing t = / (2J IS ) x H, I t / 2 t / 2 H z H z -H y a I z I z cos( J IS t) + 2I x S y sin( J IS t) S Grd Dephasing by gradients t = / (2J IS )

10 H, I t / 2 t / 2 S x ±x H z I z H y I y cos 2 ( J IS t/2) ± (-I y )sin 2 ( J IS t/2) t = / (J IS ) H z (90 x - t/2-80 x -t/2) H y I z (90 x - t/2) - I y cos( J IS t/2) + I x S z sin( J IS t/2) - I y cos( J IS t/2) + I x S z sin( J IS t/2) I y cos( J IS t/2) ± (- I x S z )sin( J IS t/2) (80 x (I) + 90 x (S) + 90 ±x (S)) ( t/2 ) I y cos( J IS t/2) ± (- I x S z )sin( J IS t/2) I y cos 2 ( J IS t/2) ± (- I y ) sin 2 ( J IS t/2)

11 Inclusion of half filters in a NOESY experiment (G. Otting, K. Wüthrich (989) J. Magn. Res. 85, ) /2 /2 /2 /2 ½ t ½ t m x 7 x dec = x, -x 2 = x 3 = 2{x}, 2{-x} 4 = 4{x}, 4{-x} 5 = 8{x}, 8{-x} 6 = x 7 = x rec = x, -x, -x, x, -x, x, x, -x -x, x, x, -x, x, -x, -x, x Four subspectra I x x II -x x III x -x IV -x -x 2

12 F F2 Subspectrum I II III IV 2 C- H - 3 C- H 2 C- H - 3 C- H 2 C- H + 3 C- H 2 C- H - 3 C- H 2 C- H - 3 C- H 2 C- H + 3 C- H 2 C- H + 3 C- H 2 C- H + 3 C- H F F2 Linear combinations (I + II) + (III + IV) (I + II) - (III + IV) (I - II) + (III - IV) (I - II) - (III - IV) 2 C- H 2 C- H 3 C- H 3 C- H 2 C- H 3 C- H 2 C- H 3 C- H

13 J( H, 3 C) Couplings in nucleic acids and proteins linear regression Nucleic acids J CH = 0.7(Hz/ppm) C + 0 Hz Proteins J CH = (Hz/ppm) C + 20 Hz

14 Problem with the variation of the J couplings x H, I / 2 / 2 S a no ideal can be found Grd = / (2J IS ) x H, I / 2 / 2 a S Evolution of the J IS -coupling: - 2 Grd = / (2J IS )

15 Filterelement with an adiabatic S inversion pulse (C. Zwahlen et al. (997), J. Am. Chem. Soc. 9, ) x H, I a a S p Amplitude [%] Phase [ ] Grd g g g Pulsdauer [us] Puls duration.948 ms (800 MHz) smoothed chirp (0 %) 40 khz sweep (low-field -> high-field)

16 Pulse sequence for a F( 3 C-filtered)-F2( 3 C-edited) NOESY -x 0 -x 4 y -y a a a a a a y y 6 p p dec g g g2 g3 g3 g4 g5 g6 g6 4 = 2{x}, 2{-x} 6 = x, -x 0 = 4{x}, 4{-x} = 8{x}, 8{-x} rec = x, -x, -x, x, -x, x, x-, -x -x, x, -x, -x, x, -x, -x, x g = 3 % g2 = 40 % g3 = 0.6 % g4 = 0. % g5 = 3 % g6 = 50 % (500 ms) ( ms) (500 ms) ( ms) ( ms) ( ms)

17 F( 3 C-filtered)-F2( 3 C-edited) NOESY of a protein-protein complex 3 C, 5 N N(-53) in complex with NusA( ) (:2) ( N ca. mm, 800 MHz, 66 h) Ausschnitte aus dem 3C editierten NOESY F(3C filtered) F2 (3C edited) NOESY H [ppm] H [ppm] 0.

18 D slices from the F( 3 C-filtered)-F2( 3 C-edited) NOESY compared with the 3 C-NOESYHSQC x 0 6 Intensität x 0 6 Intensität HD* 40.HD2* HD* 40.HD* 3 3 2D C gefiltert C editiert NOESY HSQC 3 3D C NOESY HSQC Ref. 40.HD* 4.HN Ref. 40.HD* 4.HN 40.HD2* 40.HD* 40.HD2* 40.HD2* 3 3 2D C gefiltert C editiert NOESY HSQC 3 3D C NOESY HSQC Ref. 40.HD2* 4.HN Ref. 40.HD2* 4.HN indirekte Protonendimension [ppm]