1. Resonance Assignment for Proteins Classical homonuclear ( 1 H- 1 H) assignment methods: 1. Spin system assignments 2. Sequence-specific assignments 3. Sequential vs. Main-chain Directed Assignment Modern methods: Use of heteronuclear shift correlation, triple resonance experiments, etc.

2. Resonance assignments 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 proton within some amino acid residue. Is the sharp peak at -0.8 ppm a valine, leucine or isoleucine methyl? even if we knew it was a valine methyl, which valine does it belong to? even if we knew it was Val30, which of the two methyls is it?

3. Levels of resonance assignment spin system assignment: is it Val, Ile or Leu? sequence-specific assignment: is it Val 30 or Val 87? stereospecific assignment: is it the pro-R or pro-S methyl of Val 87?

4. In “classical” protein NMR, assignments are made by using 2-dimensional experiments to establish correlations between different 1 H resonances. Recognition of characteristic patterns and networks of correlations then allows assignments to be made. Resonances are correlated either “through-bond”, mediated by the scalar coupling, or “through-space”, mediated by the spin dipolar coupling (nuclear Overhauser effect). Classical protein NMR: the basic plan HH through-space (nOe) through-bond (J-coupling)

5. 1 H chemical shift (ppm) diagonal peak: correlation of a resonance with itself crosspeak: correlation of two different resonances by short interatomic distance or through-bond connection HAHA HBHB HAHA HBHB 2 Å Basic features of 2D spectra

6. Spin systems and scalar coupling networks a spin system is a set of 1 H resonances connected (either directly or indirectly) by 1 H- 1 H scalar couplings generally this means networks of 1 H in which each 1 H is connected to another member of the network by three or fewer covalent bonds-- longer-range couplings are generally small, so experiments based on resonance correlation via scalar coupling will generally not detect four- and five-bond couplings 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 indirect connection

7. 2D COSY/TOCSY-->spin systems COSY and TOCSY give crosspeaks when resonances are linked through scalar coupling COSY gives crosspeaks when 2- and 3-bond couplings are present in TOCSY, longer range correlations are seen due to relays of 3-bond couplings these two techniques can be used to assign spin systems through recognition of coupling patterns recognition of the patterns at right also takes into account qualitative chemical shift information--the beta methyl of alanine, for instance, might be anywhere from ~0.9-1.7 but is never 3 or 4. o crosspeaks visible in COSY +, * crosspeaks visible in TOCSY

8. 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  

9. Sequence-specific assignments suppose we have the sequence of our protein from some independent measurement suppose we’ve assigned an isoleucine spin system, and there’s only one isoleucine in the sequence (unique), at position 48. Then we know our isoleucine is Ile48. there won’t be very many unique amino acid residues in a protein, however. but there will be many unique dipeptide sequences but in order to use this fact, we need to be able to connect adjacent residues. unique residues (arrows) and unique dipeptide sequences in lac repressor

10. Linking spin systems using nOe’s because the nOe depends upon interatomic distance and not upon J coupling, it can be used to connect spin systems which are adjacent in space but not part of the same spin system, for instance two residues adjacent in the sequence general nomenclature for interatomic distance between atoms A and B in residues i and j: d AB (i,j) nOe correlations are denoted using the distance nomenclature, e.g. “d  N (i,i+1) nOe” or “d  N (i,i+1) correlation” d  N (i,i+1), d NN (i,i+1), and sometimes d  N (i,i+1) are used to connect adjacent residues

11. 2D NOESY: linking spin systems 1H1H 1H1H portion of 2D NOESY of P22 cro showing d NN (i,i+1) correlations-- can “walk” along the chain from one residue to the next. Residues 3-7 shown. 3.HN/4.HN 4.HN/5.HN 5.HN/6.HN 6.HN/7.HN diagonal: no magnetization transferred crosspeaks: intersection of chemical shifts of atoms which are close in space, i.e. < 5 Å

12. Sequential assignment the technique of making the spin-system assignments, followed by sequence-specific assignment using unique fragments of sequence, is known as sequential assignment (Wuthrich) there are alternatives to this protocol: one is known as main-chain directed assignment (Englander). This technique does not focus on assigning all the spin systems first. Rather, it focuses on the backbone and links sizable stretches of backbone residues via sequential (i,i+1) nOe’s and other nOe’s that are characteristic of secondary structures. This technique is particularly useful when there is some knowledge of secondary structure beforehand.

13. Close interatomic distances in secondary structures alpha-helix parallel beta-sheet antiparallel beta-sheet type I turntype II turn

14. Close interatomic distances in 2ndary structures

15. you’ll often see nOe’s associated with secondary structure charted in this way: a thick bar means a strong nOe (short distance), a thin bar means a weak nOe (long but still visible distance) these sorts of charts allow one to make secondary structure assignments more or less concurrently with sequential assignments. As we will see, coupling constants and chemical shifts also aid in secondary structure assignment residue #

16. ...you can see that it would be easiest to link adjacent residues in helices with sequential amide-amide nOe’s, whereas in beta sheets (strand) sequential alpha-amide nOe’s are stronger d~2.8 Å d~2.2 Å

17. Arg Tyr Ser Ala Ala Asn Trp 1. assign a few unique spin systems and use as entries onto the backbone 2. walk down the backbone using sequential and other backbone nOe’s 3. fill in missing spin system assignments “backbone” refers to alpha and amide protons Summary of main-chain directed approach

18. Arg Tyr Ser Ala Ala Asn Trp 1. assign most or all spin systems 2. connect adjacent spin systems using backbone nOe’s to identify unique dipeptides 3. assemble larger sections of sequence-specific assignments from dipeptide fragments, until the whole protein has been assigned “backbone” refers to alpha and amide protons Summary of sequential approach

19. Assignment methods that use heteronuclear shift correlation for larger proteins (>10-15 kD), assignment methods based on 2D homonuclear 1 H- 1 H correlation methods (COSY/TOCSY/NOESY) don’t work very well because of overlapping resonances and broad linewidths. an alternative (which is now used even for small proteins in most cases) is to use heteronuclear shift correlation experiments on 13 C, 15 N labelled samples. in these experiments, magnetization is transferred from 1 H to 13 C and/or 15 N through large one-bond scalar couplings. Some relevant scalar coupling constants:

20. 15 N- 1 H HSQC based techniques as we have seen, one of the simplest types of heteronuclear shift correlation is the HSQC experiment, which correlates 1 H chemical shift to the chemical shift of a 15 N or 13 C connected by a single bond heteronuclear shift correlation can be combined with homonuclear experiments such as 1 H- 1 H NOESY or TOCSY to yield 3-dimensional spectra

21. 3D HSQC-NOESY and HSQC-TOCSY view of a 3D NOESY experiment these planes can be thought of as a 15 N- 1 H HSQC these planes can be thought of as a 1 H- 1 H NOESY the 15N shift dimension can resolve peaks that would overlap in a 2D NOESY

22. Triple-resonance experiments there is a whole raft of experiments that use both 13 C and 15 N correlations to 1 H nuclei the beauty of these experiments is that they can connect adjacent residues without requiring any nOe information-- it’s all through-bond scalar coupling interactions. Makes sequence-specific assignment more reliable. they also use mostly one-bond couplings, which aren’t very sensitive to the protein conformation (unlike, say, three-bond couplings, which vary significantly with conformation, as we will see) limiting factors: 13 C is expensive and these exp’ts can be tricky