Resonance assignment in proteins sequence of lysozyme: KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA KFESNFNTQATNRNTDGSTDYGILQINSRWWCN DGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVS DGNGMNAWVAWRNRCKGTDVQAWIRGCRL 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. 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?

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?

Part I: 1 H spin system assignment a spin system is a set of 1 H resonances connected (either directly or in a “relayed” fashion) by 1 H- 1 H scalar couplings (also called J couplings) generally this means networks of 1 H in which each 1 H 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. HHHH geminal coupling (two-bond) J ~ -12 to -15 Hz vicinal coupling (three-bond) J ~ 2-14 Hz HaHa HbHb HcHc example of a spin system “relayed” connection

H C N C O H C H H C H H H C N C O H CH 3 GluAla CO bonds, too far!  most residues in proteins have one 1 H 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

H C N C O H C H H C Phe C C C C C H H H H H  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 3-bond couplings 2-bond couplings

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

COSY and TOCSY spectra HHHH geminal coupling (two-bond) J ~ -12 to -15 Hz vicinal coupling (three-bond) J ~ 2-14 Hz HaHa HbHb HcHc “relayed” connections seen in TOCSY COSY is a type of 1 H- 1 H homonuclear 2D spectrum that shows crosspeaks between 1 H 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 1 H nuclei connected by more than three covalent bonds. direct two and three bond seen in COSY

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

Example of lysine spin system HH HH HH HH HH HH HH HH HH HH NH 3 + CO HN  