2 NMR ‘ journey ’ Introduction to solution NMR Alexandre Bonvin Bijvoet Center for Biomolecular Research with thanks to Dr. Klaartje Houben EMBO Global Exchange course, CCMB, Hyderabad, India November 29th - December 6th, 2012 3 Topics • Why use NMR for structural biology...? • The very basics Why use NMR.... ? • Multidimensional NMR (intro) • Resonance assignment • Structure parameters & calculations • NMR relaxation & dynamics
6 NMR & Structural biology NMR & Structural biology T R A N S I E N T D Y N A M I C S C O M P L E X E S a F helices F helices DBD CBD apo-CAP CAP-cAMP 2 Visualization of the Encounter Ensemble of the Transient Electron Transfer Complex of Cytochrome c and Cytochrome c Peroxidase Bashir Q. et al JACS (2010) Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009) 7 8 NMR & Structural biology NMR & Structural biology M E M B R A N E P R O T E I N S E X C I T E D S T AT E S Mechanisms of Proton Conduction and Gating in Influenza M2 Proton Channels from Solid-State NMR Hu F. et al Science (2010) Structure and Dynamics of Pin1 During Catalysis by NMR Labeikovsky W. et al JMB (2007)
9 10 NMR & Structural biology NMR & Structural biology A M Y L O I D F I B R I L S I N - C E L L N M R Amyloid Fibrils of the HET-s(218–289) Prion Form a β Solenoid with a High-resolution multidimensional NMR spectroscopy of proteins in human Triangular Hydrophobic Core Wasmer C. et al Science (2008) cells Inomata K. et al Nature (2009) 11 The NMR sample • isotope labeling – 15 N, 13 C, 2 H – selective labeling ( e.g. only methyl groups) – recombinant expression in E.coli The very basics of NMR • sample – pure, stable and high concentration • 500 uL of 0.5 mM solution -> ~ 5 mg per sample – preferably low salt, low pH – no additives
13 14 Nuclear spin Nuclear spin precession (rad . T -1 . s -1 ) E = µ B 0 15 16 Nuclear spin & radiowaves Boltzman distribution 1 H (I = 1/2) Larmor frequency m = - ! 1 H m = - ! ! H = " H B 0 = 2 #$ H m = ! m = !
17 18 Net magnetization Pulse B 0 B 1 " " B B ( # = # = 0 1 1 ! ! 0 2 2 1 rotating frame: observe with frequency $ 0 19 20 Chemical shielding Chemical shift shielding constant # B ( ) $ = % ! 0 1 " 2 More conveniently expressed as part per million by comparison to a reference frequency: 0 6 "# " ref ! = 1 " ref Local magnetic field is influenced by electronic environment
21 22 Free induction decay (FID) FID: analogue vs digital Free Induction Decay ( FID ) 23 24 Fourier Transform Relaxation • NMR Relaxation Signal Signal – Restores Boltzmann equilibrium FT 0 0 • T2-relaxation (spin-spin) 0 0 5 10 15 20 25 30 35 40 25 50 75 100 125 150 175 200 time (ms) freq. (s -1 ) – disappearance of transverse (x,y) magnetization – 1/T2 ~ signal line-width • T1-relaxation (spin-lattice) – build-up of longitudinal (z) magnetization FT – determines how long you should wait for the next experiment
25 26 Relaxation Relaxation Spin-lattive relaxation (restoring of equilibrium magnetization) Spin-spin relaxation (dephasing in xy plane) 1/T2 ~ signal line-width 27 28 NMR spectral quality Scalar coupling / J-coupling • Sensitivity – Signal to noise ratio (S/N) • Sample concentration • Field strength H 3 C - CH 2 - Br • .. 3 J HH • Resolution – Peak separation • Line-width (T2) • Field strength • ..
Why multidimensional NMR • 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 31 32 2D NMR 3D NMR
33 34 nD experiment Encoding information direct dimension 1D • mixing/magnetization transfer 1 FID of N points t 1 preparation acquisition indirect dimensions 2D ???? E = E = N FIDs of N points t 1 t 2 mixing preparation evolution acquisition proton A proton B 3D NxN FIDs of N points spin-spin interactions t 1 t 2 t 3 preparation evolution mixing evolution mixing acquisition 35 36 Magnetization transfer homonuclear NMR NOESY magnetic dipole t m t 1 t 2 interaction • Magnetic dipole interaction (NOE) crosspeak intensity ~1/r 6 up to 5 Å FID – Nuclear Overhauser Effect – through space – distance dependent (1/r6) COSY – NOESY -> distance restraints t 1 J-coupling interaction t 2 transfer over one J-coupling, i.e. max. 3-4 bonds FID • J-coupling interaction – through 3-4 bonds max. – chemical connectivities – assignment TOCSY J-coupling interaction t 1 t 2 – also conformation dependent transfer over several J-couplings, i.e. multiple steps over max. 3-4 mlev FID bonds
38 2D NOESY Homonuclear scalar coupling • Uses dipolar interaction (NOE) to transfer magnetization between protons – cross-peak intensity ~ 1/r 6 – distances (r) < 5Å diagonal H N 3 J H α H β ~ 3-12 Hz H N cross-peak 3 J HNH α ~ 2-10 Hz 37 39 40 2D COSY & TOCSY homonuclear NMR ~Å E = E = NOESY proton B t 2 proton A t m t 1 FID 2D COSY 2D TOCSY A ( ω A ) A ( ω A ) (F1,F2) = ω A, ω A A A H β H β B ( ω B ) (F1,F2) = ω A, ω B B ω A ω B H α H α Diagonal F1 H N H N ω A Cross-peak F2
41 42 heteronuclear NMR J coupling constants E = E = 1 J CbCg = 35 Hz 1 J CbHb = 130 Hz 1 J CaCb = 35 Hz 1 H 15 N 1 J CaC’ = 1 J NC’ = 1 J CaN = – measure frequencies of different nuclei; e.g. 1 H, 15 N, 13 C 55 Hz -15 Hz -11 Hz – no diagonal peaks 1 J CaHa = 140 Hz – mixing not possible using NOE, only via J 1 J HN = -92 Hz 2 J CaN = 7 Hz 2 J NC’ < 1 Hz 43 44 15 N HSQC 1 H- 15 N HSQC: ‘ protein fingerprint ’ – Backbone HN – Side-chain NH and NH 2
45 1 H- 15 N HSQC: ‘ protein fingerprint ’ Relaxation & dynamics 47 48 NMR relaxation Relaxation is caused by dynamics • Fluctuating magnetic fields • Return to equilibrium – Overall tumbling and local motions cause the local magnetic – Spin-lattice relaxation fields to fluctuate in time – Longitudinal relaxation → T1 relaxation B 0 • Return to z-axis B 1 – Spin-spin relaxation – Transversal relaxation → T2 relaxation • Dephasing of magnetization in the x/y plane B 0 B 0 B 1 B loc
49 50 Relaxation is caused by dynamics Local fluctuating magnetic fields • Fluctuating magnetic fields • B loc (t) = B loc [iso] + B loc (t)[aniso] – Overall tumbling and local motions cause the local magnetic fields to fluctuate in time – Isotropic part is not time dependent – B loc (t) is thus time dependent • chemical shift – If B loc (t) is fluctuating with frequency components near ω 0 then • J-coupling transitions may be induced that bring the spins back to – Only the anisotropic part is time dependent equilibrium • chemical shift anisotropy (CSA) – The efficiency of relaxation also depends on the amplitude of B loc • dipolar interaction (DD) (t) Stationary random function, B loc (t) anisotropic r 13 C interactions B loc (t) •e x B 0 t 0 CSA dipole-dipole 2 ≠ <B loc (t)> = 0 <B loc (t)> 0 What are the frequency components of B (t)? 51 52 Local fluctuating magnetic fields Components of the local field β • B loc (t)•e xy • B loc (t) = B loc [iso] + B loc (t)[aniso] non-adiabatic – Transverse fluctuating fields transitions – Isotropic part is not time dependent α – Non-adiabatic : exchange of energy between • chemical shift the spin-system and the lattice [environment] • J-coupling – Only the anisotropic part is time dependent • chemical shift anisotropy (CSA) α • dipolar interaction (DD) transitions • Only B loc (t)[aniso] can cause relaxation between states restore Boltzman – Transverse fluctuating fields: B loc (t)•e x + B loc (t)•e y β equilibrium – Longitudinal fluctuating fields: B loc (t)•e z T 1 relaxation
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