Resonance assignmentsPart 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 dipeptidesequences 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 forinteratomic distance betweenatoms A and B in residues i and j:dAB(i,j)

• nOe correlations are denoted using the distance nomenclature, e.g. “dN(i,i+1) nOe” or “dN (i,i+1) correlation”

• dN(i,i+1), dNN(i,i+1), and sometimes dN(i,i+1) are used to connect adjacent residues

fromGlasel &Deutscherp. 354

The 2D NOESY pulse sequence

mixing period m between t1 and t2 allows forcross-relaxation between nuclei(mostly zero quantum as we’ve seen)--> result is crosspeaks due to nOe

2D NOESY: linking spin systems

1H

1H

amide-amide region of 2D NOESY of P22 Cro protein, showing dNN(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: nomagnetizationtransferred

crosspeaks: intersectionof chemical shifts of atomswhich are close in space,i.e. < 5 Å

Classic 1H 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 systemsusing backbone nOe’s to identifyunique dipeptides

3. assemble larger sectionsof sequence-specific assignmentsfrom 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 uniquespin systems and useas entries onto the backbone

2. walk down the backbone usingsequential and other backbone nOe’s3. 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 antiparallelbeta-sheet

type I turn type 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 1H-1H 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 13C, 15N labelled samples.

• in these experiments, magnetization is transferred between 1H, 13C and /or 15N through large one-bond or in some cases two-bond scalar couplings.

Scalar couplings commonly used inheteronuclear shift correlation

all couplings are in units of Hz

15N-1H HSQC based techniques

•as we have seen, one of the simplest types of heteronuclear shift correlation is the HSQC experiment, which correlates 1H chemical shift to the chemical shift of a 15N or 13C connected by a single bond

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

3D HSQC-TOCSY

C

O

N

H

CH2

H

C

O

H

N

H CH3

C C

CH2

CO2-

2 of the dimensions are HN correlation (HSQC)3rd dimension is 1H-1H TOCSY correlations from the HN proton

3D HSQC-NOESY

C

O

N

H

CH2

H

C

O

H

N

H CH3

C C

CH2

CO2-

Like 3D TOCSY but includes interresidue and interspin systemcorrelations (dashed lines).

3D HSQC-NOESY and HSQC-TOCSY

view of a 3D NOESY experiment

these planes can be thought of as a 15N-1H HSQC

the planes (parallel to the slide) can be thought of as a 1H-1H NOESY

the 15N shift dimension can resolve peaks that would overlap in a 2D NOESY

HN 1H dimension

15N dimension

NOESY(1H)dimension

Analyzing 3D spectra

HN 1H dimension

15N dimension

NOESY or TOCSY(1H)dimension look at

vectors or “strips”corresponding topeaks on an HSQC(particular 15N and HNshift combinations)-->NOESY/TOCSY correlations will be alongthe length of the strip

rather than try to lookat this whole thing atonce

Extracting strips in a 3D

10 ppm

0 ppm

F1:

NOESY or TOCSY dimension

HN dimension (F3)

diagonal peak(amide region)

crosspeak toalpha

crosspeaks toside chain 1H

15N(F2in 3D)

HN (F3 in 3D)

8.1-7.9 ppm

F2 = 120 ppm(plane of paper)

F2:120 ppm, F3: 8.0 ppm

Use 2D HSQC as reference spectrum

8.1-7.9

want to look at TOCSY or NOESYcorrelations from the amide protoncorresponding to this HSQC peak

Strip of 3D corresponding to peak in HSQC

Classifying side chains in 3D TOCSY

5 ppm

0 ppm

pair of betasaround 3 ppm:aromatic (YHWF)or Asp/Asn (DN)

single pk in alpha region plus singlepeak 1-2 ppm:probable Ala (A)

set of 4 peaks in 1.9-2.6 region:Gln, Glu, Met (QEM)

5 ppm

0 ppm

5 ppm

0 ppm

(EQM) A (YHWHDN)

3D TOCSY 3D NOESY

dN(i,i+1)

dN(i,i+1)

Using 3D TOCSY/NOESY dual strip analysis

(EQM) A (YHWHDN)

same residue

different residue

TOCSY --> intraresidue xpks1. spin system classifications

NOESY --> interresidue xpks--> 2. connect strips into sequence fragments

(EQM) A (YHWFDN)

MQTLSERLKKRRIALMTQTELAVKQQSIQLIEAYVTKRPRFLFEIAMALNCDPVWLQYGTKRGKAA

only the E32-Y34 fragment matches...

4. sequence specifically assign strips in the fragment toE32, A33 and Y34.

3. take fragment from strip analysis...match (EQM)A(YHWFDN) pattern to your protein sequence...

E32 A33 Y34

15N(F2in 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 13C and 15N correlations to 1H 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: 13C is expensive and these exp’ts can be tricky

Beyond “spin systems”: connecting residues using heteronuclear J couplings

C CC

O

N

R

H

R

C

O

H

CN

R

C

O

H

N

H

-7 Hz 11 Hz

H H

the HNCA experiment above connects the HN group to the alphacarbon 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 1H-1H scalar couplings to connect residues