Filtered/edited NOESY spectra NMR Seminar HS 207 Nina Ripin 22..7
Overview NMR of biomolecular complexes Problems and Solutions Filtered/edited nomenclature Experimental elements NOESY vs filtered pulse sequence Experimental elements: Half-filter Experimental elements: Matched adiabatic inversion pulses (Nowadays) Variations of edited, filtered experiments Considerations for choosing experiments Directionality (filtered-edited vs edited-filtered) NMR NOESY spectra for protein RNA complex All-inclusive NOESY transfer NOEs
NMR of biomolecular complexes Problems: signal overlap distinguish signals from proteins and their bound ligands Solution isotope (or heteronuclear) filtering and editing selection/rejection of the magnetization which originates from protein or ligand Facilitates spectra and identification of resonances Distinguish intra-protein, intra-ligand, inter-molecular NOE cross peaks
Filtered/edited nomenclature To distinguish intra-protein, intra-ligand, inter-molecular NOE cross peaks Isotope-filtered (2D): H- 3 C (H prot ): suppressed; H- 2 C (H RNA ): intra-ligand NOEs visible H- 5 N(H prot ): suppressed; H- 4 N (H RNA ): intra-ligand NOEs visible H prot H RNA Isotope-edited (2D): H- 3 C, (H prot ): intra-protein NOEs visible H- 5 N (H prot ): intra-protein NOEs visible Isotope-resolved (3D)-> chemical shift recoded of heteronucleus unlabeled Ligand=RNA combinations of Isotope-editing, -filtering, -resolving elements inter-molecular NOEs
NOESY pulse sequence t = frequency (chemical shift) labelling of protons General Scheme for Two-Dimensional NMR scalar coupling or dipolar interaction (NOE) The pulse sequence for H- H NOESY = first scan: t short; t incremented H t t 2 Keeler; Understanding NMR Spectroscopy
What happens during the pulse sequence? During t During t m During t 2 H H H t t 2 t t 2 t t 2 H Unlabeled Ligand H 5N H 3C Protein
What happens during the pulse sequence? During t During t m During t 2 H H H t t 2 t t 2 t t 2 H Unlabeled Ligand H Unlabeled Ligand H 5N H 3C Protein NOE H 5N H 3C Protein chemical shift (CS) labelling of sender protons chemical shift (CS) labelling of receiver protons H CS - NOE - H CS 2D: All proton CS and NOEs
NOESY vs filtered/edited sequence t t 2 H H- H NOESY 2D H CS - NOE - H CS H 3 C r 3 C resolved 3D
What happens during the pulse sequence? During t During t m During t 2 3 T C r 3 H m T H H 3 C r m T C r m H Unlabeled Ligand H Unlabeled Ligand H 5N H 3C Protein NOE H 5N H 3C Protein chemical shift (CS) labelling of protons chemical shift (CS) labelling of 3 C chemical shift (CS) labelling of H H CS - NOE - 3 C CS - H CS 3D: NOEs between H- 3 C to all H
NOESY vs filtered/edited sequence t t 2 H H- H NOESY; 2D H CS - NOE - H CS t H 3 C r 3 C resolved; 3D H CS - NOE - 3 C CS - H CS t H f 3 C e 2D; +Editing- filtering elements
What happens during the pulse sequence? During t /f During t m During t 2 H f T e H f m T e H f m T e m H Unlabeled Ligand H Unlabeled Ligand H 5N H 3C Protein NOE H 5N H 3C Protein chemical shift (CS) labelling of unlabeled protons chemical shift (CS) labelling of H attached to 3 C, 5 N 2D: NOEs between unlabeled protons and protons attached to 3 C/ 5 N
NOESY vs filtered/edited sequence t t 2 H H- H NOESY; 2D t H 3 C r 3 C resolved; 3D t H f 3 C e 2D; +Editing- filtering elements Increase in length Filtering element 0.8ms ( 5 N), 6.8ms ( 3 C) transverse relaxation still active reduction of the signal Imperfect Pulses additional reduction in signal Loss in sensitivity
Experimental elements: Half-filter (older) Half-filter based on phase cycling (2 scans) Subtraction artefacts Improved half-filter: Gradient-based purging filtering in one scan (but for one scalar coupling) Matched adiabatic inversion pulses for different scalar couplings of C-H
(older) Half-filter Spin echo sequences of defined length and pulsing Refocus chemical shift ; evolve J-coupling H evolves according to scalar one-bond coupling After a time period ( st scan), H- 3 C or H- 5 N and H- 2 C or H- 4 N have opposite phases can be distinguished 2 nd scan: same phase anti-phase 2 nd scan Same phase st scan Opposite phase H- 3 C, H- 5 N H prot H- 2 C, H- 4 N H RNA Black: H RNA Blue: H prot 2 nd scan st scan H prot Visible H RNA visible
(older) Half-filter
Limitations of older Half-filters Stable spectrometer needed (vibration-free, stable temp, stable shims, lock, enough dummy scans) Otherwise subtraction will lead to artefacts work well for a single value of heteronuclear J-coupling Improved half-filter
Gradient-based purging Purge pulses or gradients De-phase H coherence Shorter (5.4ms) then normal half-filter (0.8ms) Filtering in one scan (suppressed to 0%) 2 nd scan (suppressed to %) Desired magnetization (H not coupled to 5 N) is stored along z Gradient de-phase magnetization along plane Evolution of scalar coupling J HN Black: H RNA Blue: H prot anti-phase
Nowadays: Matched adiabatic inversion pulses Other half-filters work only for one J- coupling value (as for N-H) but not the C-H couplings (Jcoupling varies for methyl -, aromatic groups, ) Adiabatic pulses for broadband inversion pulses Aim-to get uniform inversion over a large chemical shift range (inversion bandwidth) Invert 3 C nuclei in a time dependent manner (aro first and ali last) gradient-based purging to de-phase Gradient purging element Adiabatic pulse Evolution of scalar coupling Not perfect in antiphase; only partially suppressed Scalar coupling refocuses (antiphase)
Variations of isotope editing elements Isotope-editing, -filtering, -resolving elements can be combined in different ways H f r H e f H f f Filtered-edited or edited- filtered NOESY?
Variations of edited, filtered experiments
Considerations for choosing experiments Experiment depends on:. Type of NOEs wanted (intra-ligand, intra-protein, intermolecular or all together) 2. Directionality (filtered-edited vs edited-filtered) 3. Complex in fast or slow exchange on NMR time scale (next Seminar) 4. Ratio protein to ligand (transfer NOEs)
NMR NOESY spectra for protein RNA complex 3D 3 C NOESY (F 3 r) intra-protein and intermolecular NOEs 2D F f,f 2 f NOESY NOEs between unlabeled ligand 2D F f,f 2 e NOESY NOEs between unlabeled ligand + labeled proteins
NMR NOESY spectra for protein RNA complex 3D 3 C NOESY (F 3 r) intra-protein and intermolecular NOEs 2D F f,f 2 f NOESY NOEs between unlabeled ligand F 3 r F f,f 2 f NOESY
NMR NOESY spectra for protein RNA complex 3D 3 C NOESY (F 3 r) intra-protein and intermolecular NOEs 2D F f,f 2 e NOESY or 2D F e,f 2 f NOEs between unlabeled ligand + labeled proteins 2D F f,f 2 e F 3 r 2D F e,f 2 f
Filtered-edited or edited- filtered NOESY? Directionality important 2D F e,f 2 f NOESY 3D F r, F 3 f NOESY 2D F f, F 2 e NOESY 3D F f, F 3 r NOESY NOEs between H prot and H RNA NOEs between H RNA and H prot NOEs originating on protein are recorded on ligand NOEs originating on ligand are recorded on protein Same, if relaxation times T and T2 same for protein and ligand For stem loop /complex RNA: 2D F e,f 2 f, 3D F r, F 3 f (highest resolution on RNA) small RNA (easy spectrum) : 2D F f,f 2 e, 3D F f, F 3 r (highest resolution on protein) If binding weak; more ligand unbound ligand uch longer weaker NOE then for protein F e,f 2 f should be run (small ligands; short RNAs?)
Our.all Experiments NOESY-_HHC*.all NOESY-_HHN.all All in D 2 O: increase in sensitivity Filtered and edited experiments of labeled protein in complex with unlabeled ligand noesy_fff2f_d2o.all noesy_f2f_d2o.all NOESY_HfHeC_D2O.all 2D; 3C-labeled protein in complex with unlabeled ligand NOEs between unlabeled ligand 2D; 3C-labeled protein in complex with unlabeled ligand Unlabeled ligand, intermolecular NOEs 3D; 3C-labeled protein in complex with unlabeled ligand 3C-resolved F-filtered,F2-edited f = filter(detect unlabeled), e = edit(detect labeled), F=indirect dim, F2 = direct dim * Aro, Ali
Our.all Experiments Filtered and edited NOESY experiments for 3 C, 5 N labeled nucleic acid in D2O NA_noesy_FfF2f_D2O.all NA_noesy_FfF2e_D2O.all NA_noesy_FeF2e_D2O.all NA_NOESY-_HfHeC_D2O.all NA_NOESY-_HfHeCsug_D2O.all RNA assignment for partially labeled RNA RNA assignment for partially labeled RNA RNA assignment for partially labeled RNA 3 C labeled nucleic acid (in complex) 3 C sugar-only-labeled nucleic acid (in complex) f = filter(detect unlabeled), e = edit(detect labeled), F=indirect dim, F2 = direct dim
All-inclusive NOESY As normal 3D X-edited NOESY (H prot ) but includes H RNA signals in X- edited dimension intra-protein NOEs, ligand-to-protein NOEs and intra-ligand NOEs All signals in one spectra (better calibration) Ligand signals appear at 00ppm (3C dimension) More sensitive then two filtering-editing elements
Transfer NOEs (filtering based on fast exchange) Simple 2D NOESY Yield intra-ligand and intermolecular protein to ligand NOEs On small ligands in fast exchange (Kd >um) Large excess of ligand ([L]:[P] = :5-0) Only ligand visible Even if small fraction bound; positive cross peaks Intermolecular signals rather weak
Acknowledgement Thanks to Alvar! Literature Gossert et al., Structure determination of protein ligand complexes in solution; to be published A.L. Breeze Progress in Nuclear Magnetic Resonance Spectroscopy 36 (2000) 323 372