Resonance assignments Part II: Approaches to sequence-specific assignments

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 (or tripeptide etc...) 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

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

from Glasel & Deutscher p. 354 The 2D NOESY pulse sequence mixing period  m between t 1 and t 2 allows for cross-relaxation between nuclei (mostly zero quantum as we’ve seen) --> result is crosspeaks due to nOe

2D NOESY: linking spin systems 1H1H 1H1H amide-amide region of 2D NOESY of P22 Cro protein, 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

Classic 1 H resonance assignment protocols Sequential assignment method (Wuthrich) A method in which one first makes the spin-system assignments, followed by sequence-specific assignment using unique fragments of sequence. Main-chain directed assignment method (Englander). This alternative 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 (more on this in a second). This technique is particularly useful when there is some knowledge of secondary structure beforehand.

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

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

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

Close interatomic distances in 2ndary structures

nOes and secondary structures In NMR papers you’ll sometimes see charts like the one shown above. A thick bar means a strong nOe (short distance), a thin bar means a weak nOe (long but still visible distance) The fact that certain nOe’s are characteristic of secondary structures allows one to make secondary structure assignments more or less concurrently with sequential assignments. As we will soon see, coupling constants and chemical shifts also aid in secondary structure assignment residue #

...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

Modern assignment methods that use heteronuclear shift correlation for larger proteins (>10-15 kD), assignment methods based on the 2D homonuclear 1 H- 1 H correlation methods (COSY/TOCSY/NOESY) that we’ve been discussing 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 between 1 H, 13 C and /or 15 N through large one-bond or in some cases two-bond scalar couplings.

Scalar couplings commonly used in heteronuclear shift correlation all couplings are in units of Hz

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 2D heteronuclear shift correlation can be combined with homonuclear experiments such as 1 H- 1 H 2D NOESY or 2D TOCSY to yield 3-dimensional spectra

3D HSQC-TOCSY C O N H CH 2 H C O H N H CH 3 CC CH 2 CO of the dimensions are HN correlation (HSQC) 3rd dimension is 1 H- 1 H TOCSY correlations from the HN proton

3D HSQC-NOESY C O N H CH 2 H C O H N H CH 3 CC CH 2 CO 2 - Like 3D TOCSY but includes interresidue and interspin system correlations (dashed lines).

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 the planes (parallel to the slide) can be thought of as a 1 H- 1 H NOESY the 15 N shift dimension can resolve peaks that would overlap in a 2D NOESY HN 1 H dimension 15 N dimension NOESY ( 1 H) dimension

Analyzing 3D spectra HN 1 H dimension 15 N dimension NOESY or TOCSY ( 1 H) dimension look at vectors or “strips” corresponding to peaks on an HSQC (particular 15N and HN shift combinations)--> NOESY/TOCSY correlations will be along the length of the strip rather than try to look at this whole thing at once

Extracting strips in a 3D 10 ppm 0 ppm F1: NOESY or TOCSY dimension HN dimension (F3) diagonal peak (amide region) crosspeak to alpha crosspeaks to side chain 1 H 15 N (F2 in 3D) HN (F3 in 3D) ppm F2 = 120 ppm (plane of paper) F2:120 ppm, F3: 8.0 ppm Use 2D HSQC as reference spectrum want to look at TOCSY or NOESY correlations from the amide proton corresponding to this HSQC peak Strip of 3D corresponding to peak in HSQC

Classifying side chains in 3D TOCSY       5 ppm 0 ppm pair of betas around 3 ppm: aromatic (YHWF) or Asp/Asn (DN)  single pk in alpha region plus single peak 1-2 ppm: probable Ala (A) set of 4 peaks in region: Gln, Glu, Met (QEM) 5 ppm 0 ppm 5 ppm 0 ppm

(EQM)A (YHWHDN) 3D TOCSY 3D NOESY d  N (i,i+1) d  N (i,i+1)       Using 3D TOCSY/NOESY dual strip analysis (EQM)A(YHWHDN) same residue different residue TOCSY --> intraresidue xpks 1. spin system classifications NOESY --> interresidue xpks--> 2. connect strips into sequence fragments

(EQM)A(YHWFDN) MQTLSERLKKRRIALMTQTELAVKQQ SIQLIEAYVTKRPRFLFEIAMALNCDPV WLQYGTKRGKAA only the E32-Y34 fragment matches... 4.sequence specifically assign strips in the fragment to E32, A33 and Y take fragment from strip analysis...match (EQM)A(YHWFDN) pattern to your protein sequence... E32A33Y34

15 N (F2 in 3D) HN (F3 in 3D) E32 A33 Y34 5. annotate the corresponding 2D HSQC peaks with the new assignments 6. proceed until entire HSQC is assigned...

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

Beyond “spin systems”: connecting residues using heteronuclear J couplings CC C O N R H R C O H CN R C O H N H -7 Hz11 Hz HH the HNCA experiment above connects the HN group to the alpha carbon of both the same residue and the previous one. The two-bond N-C coupling traverses the carbonyl group, which is a barrier to using 1 H- 1 H scalar couplings to connect residues