1 / 15

Protein

Protein. NMR. Part II. 1. Protein Structures by NMR. NMR, UNLIKE Xray crystallography and EM, DOES NOT experimentally produce a protein structure. NMR yields Distance Restraints , which are used to CALCULATE protein structures. NMR structure calculations yield

lars
Download Presentation

Protein

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Protein NMR Part II

  2. 1. Protein Structures by NMR NMR, UNLIKE Xray crystallography and EM, DOES NOT experimentally produce a protein structure. NMR yields Distance Restraints, which are used to CALCULATE protein structures. NMR structure calculations yield MULTIPLE solutions (ensembles). NMR protein structures are always ENSEMBLE AVERAGES.

  3. 2. NMR Structure Calculation Long-Range/Short-Range Distance Restraints H3 Lys10 H1 Ser57 H2 Asp11 H3 Lys72 H2 Arg98 > Int. 0.75 H2 Glu42 > Int. 0.45 H2 Arg98 > Int. 0.55 H2 Arg98 > Int. 0.95 Energy Minimization Short-Range > 2nd. Structure Long-Range > Tertiary Structure (Protein Fold) Assessment of Structural Quality r.m.s.d., Ramachandran Plot etc.

  4. 3. The Nuclear Overhauser Effect (NOE Experiments) Proton-Proton Distances

  5. 4. Problems with Proteins 1H 1H 1H 1H 15N Spectral Overlap Spectral Editing (2D or 3D) Selective Labeling

  6. 5. Protein NOEs -sheet NOEs -helical NOEs

  7. 6. … yet, how do we know which one is which? Goal: Identify the Resonance Frequencies of ALL Proton, Carbon and Nitrogen nuclei in a protein. Backbone Assignment Strategies Side-Chain Assignment Strategies

  8. 7. Magnetization Transfer i 1H/15N Correlation (2D) HSQC or HMQC-type 15N i (-1) 1H Magnetization transfer through space > NOE Magnetization transfer through bonds > J-coupling HNCA Experiment (3D) HNCACB Experiment (3D)

  9. 8. Assignment C i C i (-1) C i (-1) i C i i (-1) HNCACB Experiment (as an example) What have we learned? The Amide Proton resonance frequency The Amide Nitrogen resonance frequency The AlphaCarbon resonance frequency The BetaCarbon resonance frequency i (-1) i

  10. 9. NMR Experiments Types of experiments and nomenclature BACKBONE EXPERIMENTS HNCA HN(CO)CA HN(CA)CO HNCO HNCACB HN(CO)CACB

  11. 9. NMR Experiments (cont.) Types of experiments and nomenclature SIDE-CHAIN EXPERIMENTS (H)CC(CO)NH (H)CCNH H(CC)NH H(CC)(CO)NH HCCH-COSY HCCH-TOCSY

  12. 10. Putting things together … Hence: A 3D HCCH-TOCSY looks like a 3D 13C-edited NOESY without NOE Cross-peaks.

  13. 11. NMR Pulse-Sequences How do we READ them? What do they MEAN and how do they WORK?

  14. 12. Let’s do one together … Can you point outthe basic BUILDING BLOCKS? Can you determine the MAGNETIZATION TRANSFER PATH? Can you identify the NMR EXPERIMENT?

  15. 13. Summary NMR structures are COMPUTED and not experimentally determined. To calculate an NMR structure we need DISTANCE RESTRAINTS. Distance restraints are provided by NOE EXPERIMENTS. NOE experiments are SPECTRAL EDITED and recorded in 3D. NOE experiments are meaningless unless we have a COMPLETE NMR RESONANCE ASSIGNMENT. Resonance assignments are given by BACKBONE- and SIDE-CHAIN NMR EXPERIMENTS (which exploit J-COUPLINGS). Backbone- and side-chain experiments are recorded as sets of 3D EXPERIMENTS employing tailored NMR PULSE SEQUENCES. NMR pulse-sequences are made up of specifically arranged BUILDING BLOCKS .

More Related