1 / 9

Resonance assignment in proteins

Resonance assignment in proteins. in order to be able to actually solve the structure of a protein, we first have to assign the spectrum. Each peak corresponds to some 1 H nucleus within some amino acid residue. The sharp peak at -0.8 ppm is almost certainly a methyl.

willa
Download Presentation

Resonance assignment in proteins

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. Resonance assignment in proteins • in order to be able to actually solve the structure of a protein, we first have to assign the spectrum. Each peak corresponds to some 1H nucleus within some amino acid residue. • The sharp peak at -0.8 ppm is almost certainly a methyl. • Is it a valine, leucine or isoleucine methyl? • suppose we knew it was a valine methyl. We still don’t know: • Which valine in the protein does it belong to? • suppose we knew it was Val30. We still don’t know • Which of the two methyls of Val30 is it? sequence of lysozyme: KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL

  2. Levels of resonance assignment • residue or “spin system” assignment: is it a Val, Ile or Leu methyl? • sequence-specific assignment: is it in Val 30 or Val 87? • stereospecific assignment: is it the pro-R or pro-S methyl of Val 87?

  3. Part I: 1H spin system assignment • a spin system is a set of 1H resonances connected (either directly or in a “relayed” fashion) by 1H-1H scalar couplings (also called J couplings) • generally this means networks of 1H in which each 1H is connected to some other member of the network by three or fewer covalent bonds--The reason for defining it this way is that four and five bond couplings are generally too small (<1 Hz) to be seen in NMR experiments. Hc “relayed” connection H H H H Ha Hb geminal coupling (two-bond) J ~ -12 to -15 Hz vicinal coupling (three-bond) J ~ 2-14 Hz example of a spin system

  4. most residues in proteins have one1H spin system • spin systems never span more than a single residue--the carbonyl group assures that the closest coupling between residues is never fewer than four bonds... 3-bond couplings 2-bond couplings CO2- H C H H CH3 C H H H 4-bonds, too far! C C C C N N H O H O Glu Ala

  5. H 3-bond couplings 2-bond couplings C H H C C C C H H C C H H H C • Some residue types contain multiple spin systems • for example, phenylalanine has a separate spin system for the aromatic ring and for the amide-alpha-beta linkage C N H O Phe

  6. Homonuclear J-coupled 2D spectra scalar or J-coupled protons HA HB HA HB HA diagonal peak: correlation of a resonance with itself 1H chemical shift (ppm) crosspeak: correlation of two different resonances by through-bond scalar coupling connection HB 1H chemical shift (ppm)

  7. COSY and TOCSY spectra COSY is a type of 1H-1H homonuclear 2D spectrum that shows crosspeaks between 1H nuclei with geminal or vicinal coupling (two or three bond) TOCSY is similar but includes “relays” of coupling so that crosspeaks can be observed between 1H nuclei connected by more than three covalent bonds. direct two and three bond seen in COSY “relayed” connections seen in TOCSY Hc H H H H geminal coupling (two-bond) J ~ -12 to -15 Hz vicinal coupling (three-bond) J ~ 2-14 Hz Ha Hb

  8. 2D COSY and TOCSY-->spin systems • COSY and TOCSY can be used to assign spin systems through recognition of coupling patterns • important to note that recognition of the patterns at right also takes into account qualitative chemical shift information--for example, Gly and Ala are distinguished from each other by the fact that the coupled resonances of Gly are both in the alpha region (3-5.5), while for Ala we see a coupling between resonances in the alpha region and in the methyl region (0-1.7). So the Ala crosspeak will be much further from the diagonal (see figure). o crosspeaks visible in COSY +, * crosspeaks visible in TOCSY

  9. Example of lysine spin system CO Hd HN NH3+ Ha He Hb Hg e a b g d Hg Hd Hb He Ha

More Related