1 Resonance assignment strategies

2 Amino acid sequence + The assignment problem

3 Assignment via 1 H NMR

4 Proton frequencies

- Assignment based on backbone H N ➡ present in all residues (except Proline) ➡ unique region of spectrum ➡ well-dispersed resonances - Scalar couplings (COSY / TOCSY) ➡ identify spin systems (i.e. amino acid type) - number of resonances (i.e. protons) -frequency of resonances -Connect with NOESY Spin Systems

6 Dipolar interaction (NOEs) ➡ through-space contacts ➡ intra-residual, sequential (& long-range) contacts ➡ link spin-systems: identify i & i-1 residue "i -1"residue "i" 1 H - 1 H NOE intra-residual NOEs sequential NOEs residue "i+1"

7 Peptide sequence R1-A2-Q3-L4-A5-M6-S7 Example Intra residue Inter residue

8 Example: who is who? Intra residue Inter residue ??? Peptide sequence R1-A2-Q3-L4-A5-M6-S

9 Example: identify protons & frequencies Intra residue Inter residue Peptide sequence R1-A2-Q3-L4-A5-M6-S

10 Example: assign strips to residues Intra residue Inter residue C = Ala Peptide sequence R1-A2-Q3-L4-A5-M6-S

11 Example: assign strips to residues Intra residue Inter residue C = Ala B = Leu Peptide sequence R1-A2-Q3-L4-A5-M6-S

12 Example: assign strips to sequence Intra residue Inter residue C = A2 / A5 B = L4 A = Q3 / M6 Possibilities I: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC) III: L4 - A5 - M6 (BCA) Peptide sequence R1-A2-Q3-L4-A5-M6-S

13 Example: connect residues Use H N -H N NOEs ➡ B has cross-peaks to both A & C ➡ ABC ➡ Q3 - L4 - A5 Peptide sequence R1-A2-Q3-L4-A5-M6-S7 Possibilities I: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC) III: L4 - A5 - M6 (BCA)

14 Example: verify!!! Use H N -H α NOEs to verify ➡ sequential H N (i) - H α (i-1)  H N (C) - H α (B)  H N (B) - H α (A) ➡ ABC ➡ Q3 - L4 - A5 Peptide sequence R1-A2-Q3-L4-A5-M6-S7 Possibilities I: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC) III: L4 - A5 - M6 (BCA)

15 Assignment via 1 H, 15 N, and 13 C Assignment via 1 H, 15 N, and 13 C

J coupling constants

17 Heteronuclear experiments ➡ more information ➡ increase resolution: 2D → 3D → 4D... ➡ sequential assignment based on scalar coupling Triple resonance NMR Advantages ➡ through-bond (J) magnetization transfer to neighboring residues (instead of NOE) ➡ 1 J scalar coupling much larger than 3 J HH (<10 Hz) (efficient transfer of magnetization) Protons Other nuclei 13 C, 15 N

18 Nomenclature Names of scalar experiments based on atoms detected HNCA HN(CO)CA HN(CA)CO HNCO HN(CA)CB HN(COCA)CB Pairs of experiments distinguish between intra-residual and sequential resonances Residuei-1 & ii-1i-1 & ii-1 i-1 & ii-1

Example: 3D HNCA

20 Example: analyze frequencies a b N– 13 C α – 13 C– 15 N– 13 C α – 13 C-- H R H R HH OO H R H R HH OO c N– 13 C α – 13 C– 15 N– 13 C α – 13 C-- H R H R HH OO 61.32

21 Numerically... ➡ c: C α (i) = a: C α (i-1) ➡ a: C α (i) = b: C α (i-1) Example: link the spin-systems Sequence: c – a – b

Example: link the spin-systems

23 13 C α 1HN1HN 15 N i-1 i & i-1 HNCA versus HN(CO)CA

24 Assigned [ 1 H- 15 N]-HSQC 15 N 1H1H

25 If no label or only 15 N: NOESY / TOCSY Identify spin-system in TOCSY Sequential NOEs to link spin-systems 13 C & 15 N: 3D triple resonance experiments Sequential information through bond (J coupling) HNCA / HN(CO)CA (and many more) Key concepts assignment

/81 26 NMR observables & structural restraints

/81 27 Protein structure Secondary structure  alpha helix, beta-sheet, etc. Tertiary structure  full 3D structure Experimental data that give information about the protein structure  NMR observables Translate the experimental data into parameters that can be used in a structure calculation  Structural restraints

/81 28 NMR observables vs. structural restraints - 3 J-couplingdihedral angle - Chemical shiftssecondary structure - NOE’sH-H distances - Paramagnetic relaxation enhancement (PRE)distances - Residual dipolar coupling (RDC)orientation of vectors - H/D exchangehydrogen bonds

/81 29 Karplus relation: J = A.cos 2 (φ) + B.cos (φ) + C measured 3 J(H N H α ) reports on φ φ OBSERVABLE: homonuclear J-couplings φ

/81 30 φ ω ~ 180º NN CC C C C C OO ψω RESTRAINTS: dihedral angles

/ C α and 13 C β chemical shifts  sensitive to dihedral angles  report on secondary structure elements OBSERVABLE: chemical shift

/81 Chemical Shift Index (CSI)

/81 Predicting dihedral angels: TALOS

/81 34 anti-parallel β-strandα-helix φ ψ β-strand α-helix RESTRAINTS: dihedral angles φψ ψ φ

/ ψ φ +180 α-helix β-strand Ramachandran plot

/ H- 1 H NOEs  signal intensity proportional to 1/r 6  reports on distance between protons ➡ distance restraints (up to 5-6 Å) Sequential ABC D Z Intra-residue ( used for identifying spin-systems ) Medium range Sequential & medium range NOEs - SECONDARY STRUCTURE OBSERVABLE: NOE Longe range r =

/81 37 RESTRAINT: distances

/81 38 NOEs in secondary structure elements

/81 39 Short distances in β-strands anti-parallel

/81 40 NOEs in secondary structure elements

/81 41 Short distances in α-helices

/81 42 Short distances in α-helices

/81 43 OBSERVABLE: PRE paramagnetic relaxation enhancement (PRE) paramagnetic center (unpaired electron)  radical (e.g. nitroxide)  certain metal ions (i.e. Mn 2+, Gd 3+ ) nuclear spin relaxation is enhanced by the paramagnetic center  signals will broaden (or even disappear)  effect is dependent on the distance to the paramagnetic center ➡ 1/r 6  because of the large magnetic moment of the unpaired electron the PRE provides long-range distance information (Mn 2+ ~35 Å)

/81 OBSERVABLE: Residual dipolar couplings Dipolar coupling

/81 Residual dipolar coupling (RDC) Dipolar coupling

/81 Residual dipolar coupling (RDC)

/81 RDC reports on orientation of bond-vector - orientation of bond-vector within a molecular alignment tensor (defined by A a and A r ) with respect to the magnetic field Long range orientational restraint - TERTIARY STRUCTURE RDC: Orientational restraint

/81 48 The more RDCs, the better... NN CC C C C C OO RDCs commonly measured  1 D 1 H N - 15 N  1 D 13 C’- 15 N  1 D 13 C α - 13 C’  1 D 1 H α - 13 C α In perdeuterated proteins  2 D 1 H N - 13 C’  2 D/ 3 D 1 H N - 13 C α

/81 49 RESTRAINT: RDC Orientation

/81 OBSERVABLE: H/D exchange rates

/81 51 OBSERVABLES  chemical shifts ( 1 H, 15 N, 13 C,...)  J-couplings, e.g. 3 J(H N,H α )  medium-range NOEs  hydrogen/deuterium exchange Sources of structural information  long-range NOEs  residual dipolar couplings (RDCs)  paramagnetic relaxation enhancement (PREs) Secondary structure Tertiary structure