Introduction solution NMR

2 NMR journey Introduction solution NMR Alexandre Bonvin Bijvoet Center for Biomolecular Research with thanks to Dr. Klaartje Houben EMBO Global Exchange course, IHEP, Beijing April 28 - May 5, 20 3 Topics Why use NMR for structural biology...? The very basics Multidimensional NMR Why use NMR...? Resonance assignment Structural parameters NMR relaxation & dynamics

NATURE Vol 462 9 November 2009 NMR & Structural biology LETTERS 5 NMR & Structural biology 6 F helices F helices DBD WT-CAP CBD apo-cap CAP-cAMP 2 CAP-cAMP 2 -DNA b c 0.6 Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009) Figure Conformational states of CAP and effect of camp binding assessed by NMR. a, Structures of CAP in three ligation states: apo 9, camp2-bound 0, and camp2-dna-bound 8. The CBD, DBD and hinge region are coloured blue, magenta and yellow, respectively. camp and DNA are displayed as grey and green sticks, respectively. b, c, Effect of camp Δ! (p.p.m.) binding on the structure of WT-CAP (b) and CAP-S62F (c) as assessed by...allows to chemical study shift mapping (Supplementary the Fig. dynamics S4). Chemical shift difference of (Dv; p.p.m.) values are mapped by continuous-scale colour onto the WT- CAP-cAMP2 structure. biomolecular systems... Δ! (p.p.m.) 0.0 High-resolution multidimensional NMR spectroscopy of proteins in human cells Inomata K. et al Nature (2009)...structural studies in membrane and whole cells possible! The Sample isotope labeling 369 unlabeled 2009 (peptides) Macmillan Publishers Limited. All rights reserved 5 N labelled (small proteins < 0 kda) 5 N & 3 C labelled (larger proteins, up to 30-40 kda) 5 N, 3 C & 2 H labelled (large proteins > 40 kda) protein production (E.coli) quite a lot & very pure & stable 500 ul of 0.5 mm solution -> ~ 5 mg per sample 3 C labelling is costly, ~k! per sample preferably low salt, low ph, no additives. 7 Pros & cons of solution NMR in structural biology Pros... no need for crystal: no crystal packing artefacts, solution more native-like potential to study dynamics: picosecond to seconds time scales, conformational averaging, chemical reactions, folding... easy study of protein-protein, protein-dna, proteinligand interactions Cons... NMR structure determination is a bit slow... Need isotope labeling ( 3 C, 5 N) solution NMR works best for MW < 50 kda 8

0 The very basics of NMR Nuclear spin precession Nuclear spin 2 (rad. T -. s - ) E = µ B 0

Larmor frequency H (I = /2) m = -! Larmor frequency! H = " HB 0 = 2#$ H 3 4 Boltzman distribution H (I = /2) m = -! m =! m =! Net magnetization 5 Chemical shielding Local magnetic field is influenced by electronic environment 6

Chemical shielding # B0 $ = % 2" (! ) Chemical shielding 7 Chemical shift Chemical shift: δσiso [Hz] = νobs - ν0 8 δσiso [ppm] = (νobs - ν0)/(ν0. 0-6 ) ppm: parts per million ppm value is not field dependent 4 Tesla:!H = 600 MHz ppm = 600 Hz ( H) 2 Tesla:!H = 900 MHz ppm = 900 Hz ( H) Pulse 9 FID: analogue vs digital 20 Observe with the Lamor frequency rotating frame Free Induction Decay (FID)

2 22 Fourier Transform Relaxation Signal 0 FT Signal 0 NMR Relaxation Restoring Boltzmann equilibrium 0 25 50 75 00 25 50 75 200 time (ms) 0 5 0 5 20 25 30 35 40 freq. (s - ) T2-relaxation disappearance of transverse (x,y) magnetization /T2 ~ signal line-width FT T-relaxation build-up of longitudinal (z) magnetization determines how long you should wait for the next experiment 23 24 NMR spectral quality Scalar coupling / J-coupling Sensitivity Signal to noise ratio (S/N) Sample concentration Field strength.. Resolution Peak separation Line-width (T2) Field strength.. H3C - CH2 - Br 3 JHH

Why multidimensional NMR 26 multidimensional NMR experiments resolve overlapping signals enables assignment of all signals Multidimensional NMR encode structural and/or dynamical information enables structure determination enables study of dynamics 2D NMR 27 3D NMR 28

29 30 D nd experiment direct dimension t single FID of N points Encoding information mixing/magnetization transfer FID indirect dimensions 2D t mixing FID t2 N FIDs of N points???? 3D t mixing t2 mixing FID t3 NxN FIDs of N points proton A spin-spin interactions proton B 3 32 Magnetization transfer homonuclear NMR magnetic dipole interaction (NOE) Nuclear Overhauser Effect through space distance dependent (/r6) NOESY -> distance restraints J-coupling interaction through 3-4 bonds max. chemical connectivities assignment also conformation dependent NOESY COSY TOCSY t t tm t mlev FID FID FID t2 t2 t2 magnetic dipole interaction crosspeak intensity ~/r 6 up to 5 Å J-coupling interaction transfer over one J-coupling, i.e. max. 3-4 bonds J-coupling interaction transfer over several J-couplings, i.e. multiple steps over max. 3-4 bonds

33 34 homonuclear NMR ~Å 2D COSY & TOCSY NOESY t tm FID t2 proton A proton B 2D COSY 2D TOCSY A A (ωa) A A (ωa) (F,F2) = ωa, ωa B B (ωb) (F,F2) = ωa, ωb Hβ Hβ ωa ωb Hα Hα Diagonal F ωa Cross-peak HN HN F2 35 36 heteronuclear NMR J coupling constants J CbCg = 35 Hz J CbHb = 30 Hz H 5 N J CaCb = 35 Hz measure frequencies of different nuclei; e.g. H, 5 N, 3 C no diagonal peaks mixing not possible using NOE, only via J J CaC = 55 Hz J NC = J CaN = -5 Hz - Hz J CaHa = 40 Hz 2 J CaN = 7 Hz J HN = -92 Hz 2 J NC < Hz

37 38 J coupling constants heteronuclear NMR HSQC (heteronuclear single quantum coherence) H 5 N J-mix block t J-mix block t2 FID DEC JNH H 5 N (ω N) 5 JNH H (ω H) (F,F2) = ω 5 N, ω H J HN = -92 Hz 39 40 H- 5 N HSQC: protein fingerprint H- 5 N HSQC: protein fingerprint note that spectrum is decoupled: no NH J- coupling

4 42 J coupling constants Triple resonance NMR HNCA H J-mix block J-mix block FID t3 J CaN = - Hz 5 N 3 C J-mix block t J-mix block t2 DEC 2J CaN = 7 Hz H JNH 5 JNCa(i) N 2 JNCa(i-) JNCa(i) 3 C (ω C) 5 N (ω N) H (ω H) 3 5 2 JNCa(i-) JNH J HN = -92 Hz (F,F2,F3)= (ω 3 Ca(i), ω 5 N(i), ω H(i)) & (ω 3 Ca(i-), ω 5 N(i), ω H(i)) Resonance assignment Structural parameters

Structural study by NMR Sample preparation (months) Acquisition of NMR spectra (~ month) Sources of structural information OBSERVABLES chemical shifts ( H, 5 N, 3 C, 3 P) J-couplings, e.g. J(HN,Hα) Secondary structure Chemical shift assignments Backbone (days) Side-chains (days) Analysis of NOESY spectra (weeks) Structure calculations (days) Functional studies with NMR Interaction with partner a few months or more... long-range NOEs residual dipolar couplings H / D exchange effects of ph / T effects of interacting partners relaxation rates... Tertiary structure RESTRAINTS! dihedral angles! distances between atoms! orientation between bond vectors 47 48 RESTRAINTS: dihedral angles RESTRAINTS: dihedral angles ω ~ 80º anti-parallel β-strand α-helix C C N C C N C C ψ φ ψ O ω O φ ψ φ φ φ

49 50 Ramachandran plot OBSERVABLE: chemical shift +80 β-strand 3 Cα and 3 Cβ chemical shifts sensitive to dihedral angles report on secondary structure elements β-strand α-helix φ -30-60 ψ 25-45 ψ -80 α-helix -80 φ +80 OBSERVABLE: homonuclear J- couplings 5 RESTRAINT: distances 52 φ! φ! Karplus J = A.cos 2 (φ) + B.cos (φ) + C measured 3 J(HNHα) reports on φ

53 54 OBSERVABLE: NOE H- H NOEs (2D NOESY, 3D NOESY-HSQC) signal intensity proportional to /r 6 reports on distance between protons distance restraints OBSERVABLE: NOE H- H NOEs signal intensity proportional to /r 6 reports on distance between protons distance restraints r = r = Sequential H x H y Cross-peak between H x and H y Intra-residue (used for identifying spin-systems) A B C D Z Medium range Sequential & medium range NOEs - SECONDARY STRUCTURE NOEs in secondary structure elements 55 OBSERVABLE: NOE 56 H- H NOES signal intensity proportional to /r 6 reports on distance between protons distance restraints Longe range Sequential Intra-residue (used for identifying spin-systems) A B C D Z Medium range Long range NOEs - TERTIARY STRUCTURE

57 58 Long-range NOEs RESTRAINT: Orientation Tertiary information distances < 5 Å Important structural information anti-parallel OBSERVABLE: Residual dipolar couplings Dipolar coupling D ij = " # i# j!µ 0 4$ 2 r 3 3cos 2 %(t) " 2 59 OBSERVABLE: Residual dipolar couplings Dipolar coupling D ij = " # i# j!µ 0 4$ 2 r 3 3cos 2 %(t) " 2 60 B 0 B 0 Ω No protein alignment ISOTROPIC SYSTEM D = 0 No protein alignment ISOTROPIC SYSTEM D = 0 Protein alignment ANISOTROPIC SYSTEM D 0

OBSERVABLE: Residual dipolar couplings ISOTROPIC JNH 6 Residual dipolar coupling RDC reports on orientation of bond-vector orientation of bond-vector within an alignment tensor (defined by Aa and Ar) with respect to the magnetic field i.e. orientation of bond vector with respect to other bonds 62 JNH JNH + DNH B ANISOTROPIC Difference gives RDC DNH D ij = " # i# j!µ 0 ' A 8$ 2 r 3 a (3cos 2 % ") + 3 2 A * r sin 2 % cos(2&) ( ) +, Long range orientational restraint - TERTIARY STRUCTURE OBSERVABLE: H/D exchange rates 63 OBSERVABLE: chemical shift changes 64 Exchange H by D ( 2 H) peaks disappear in time accessibility of sites stability of secondary structure elements H-bonds!2 k open # # $ N! H " "" O = C N! H k int ## $ N! D k % # close # Titration add in steps an interacting molecule (ligand / protein / DNA) observe changes in chemical shift map interaction site 06.0.0! N5 6.0 increasing H N protection!3 2.0 26.0 9.0 8.0 H 7.0

OBSERVABLE: chemical shift changes Titration add in steps an interacting molecule (ligand / protein / DNA) observe changes in chemical shift map interaction site 65 Key concepts structural parameters OBSERVABLES chemical shifts ( H, 5 N, 3 C, 3 P) J-couplings, e.g. 3 J(H N,H α ) medium-range NOEs long-range NOEs residual dipolar couplings (RDCs) RESTRAINTS dihedral angles dihedral angles medium range distances long-range distances orientations bond-vectors 66 H / D exchange effects of interacting partners... accessibility / H-bonds interaction surface... NMR time scales 68 Relaxation & dynamics protein dynamics protein folding domain motions loop motions side chain motions bond vibrations overall tumbling enzyme catalysis; allosterics fs ps ns s ms s NMR R,R 2,NOE relaxation dispersion real time NMR J-couplings H/D exchange RDC

69 70 Local fluctuating magnetic fields Relaxation Fast motion Locally induced magnetic field changes Causes relaxation Return to equilibrium Longitudinal relaxation T relaxation Return to z-axis B0 B B0 Transversal relaxation T2 relaxation Dephasing of magnetization in the x/y plane B0 B Binduced Relaxation 7 FAST (ps-ns): rotation correlation time 72 relaxation time is related to rate of motion R = /T R2 = /T2

5 FAST (ps-ns): protein flexibility 0 20 5 0 R (s-) 73 NMR time scales 74 (b) 5 0 5 R /R R2/R protein dynamics protein folding domain motions loop motions side chain motions bond vibrations overall tumbling enzyme catalysis; allosterics 27 237 57 77 97 27 237 mber residue number C tail 0 3 loop N NMR fs ps ns s ms s R,R 2,NOE RDC relaxation dispersion J-couplings real time NMR H/D exchange 7 SLOW (µs-ms): conformational exchange 75 SLOW (µs-ms): conformational exchange 76 6 0 (ppm) Causes line-broadening Makes T2 relaxation faster

Key concepts relaxation 77 wide range of time scales fluctuating magnetic fields rotational correlation time (ns) fast time scale flexibility (ps-ns) slow time scale (μs-ms): conformational exchange The End Thank you for your attention!