The NMR research group Prof. Marc Baldus Prof. Rolf Boelens Dr. - PowerPoint PPT Presentation

1 Introduc=on to solu=on NMR Alexandre Bonvin Bijvoet Center for Biomolecular Research Utrecht University, the Netherlands The NMR research group Prof. Marc Baldus Prof. Rolf Boelens Dr. Hugo van Ingen Solution NMR: 950-cryo, 900-cryo, 750, 600-cryo, 600US, 2x500 MHz 2020? 1.2 GHz Solid-state NMR: 800WB-DNP, 400WB-DNP, 700US, 500WB MHz Dr. Markus Weingarth e-infrastructure: >1900 CPU cores + EGI grid (>100’000 CPU cores) Prof. Alexandre Bonvin National and European infrastructure http://www.uu.nl/nmr

5 6 Topics NMR ‘ journey ’ • Why use NMR for structural biology...? • The very basics • Multidimensional NMR (intro) • Resonance assignment (lecture Banci) • Structure parameters & calculations (lecture Banci) • NMR relaxation & dynamics NMR & Structural biology D Y N A M I C S a F helices F helices Why use NMR.... ? DBD CBD apo-CAP CAP-cAMP 2 Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009)

NMR & Structural biology NMR & Structural biology D Y N A M I C S B i o m o l e c u l a r i n t e r a c t i o n s • Allosteric regulation • Even weak and transient complexes can be studied • Dynamic interaction between ligand-binding & DNA binding site Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009) 11 12 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 • Native like environment • Structural changes due to lipid environment Shekhar & Kay PNAS 2013 van der Cruijsen, ..... & Baldus PNAS 2013

13 14 NMR & Structural biology NMR & Structural biology I N - C E L L N M R A M Y L O I D F I B R I L S • Study proteins in their native cellular environment • Outermembrane protein in bacterial cell envelop Amyloid Fibrils of the HET-s(218–289) Prion Form a β Solenoid with a Triangular Hydrophobic Core Wasmer C. et al Science (2008) Renault M, ..... & Baldus PNAS 2012 16 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

17 18 Nuclear spin Nuclear spin precession (rad . T -1 . s -1 ) E = µ B 0 19 20 Nuclear spin & radiowaves Nuclear spin • Nuclear magnetic resonance • NMR a non invasive technique • Only nuclei with non-zero spin quantum number are “ magnets ” • Low energy radiowaves • Commonly used spins are spin ½ nuclei: 1 H, 13 C, 15 N, 31 P etc . quantum number I = ½ magnetic field strength β - ½ Δ E = γħ B 0 B 0 α β ½ α gyromagnetic ratio (different for each type of nucleus) Larmor frequency ν = ( γ B 0 )/2 π

21 22 Boltzman distribution Net magnetization 1 H m = - ½ m = ½ Example - 20.001 spins - Only 1 more spin in lower energy state 24 Pulse Chemical shielding • Radio frequency pulses • Turn on an amplifier for a certain amount of time & certain amount of power (B 1 field) γ B ( ν = 0 1 B 0 π 0 2 only rotation B 1 around B 1 is γ B ν = observed 1 π 2 1 Local magnetic field is influenced by electronic rotating frame: observe with frequency ν 0 environment ==> frequencies of nuclei will differ

25 26 Chemical shift The spectrometer shielding constant γ B ( ) ν = − σ 0 1 π 2 More conveniently expressed as part per million by comparison to a reference frequency: 0 6 "# " ref ! = 1 " ref 27 28 Free induction decay (FID) FID: analogue vs digital Free Induction Decay ( FID )

29 30 Relaxation Fourier Transform • NMR Relaxation Signal Signal – Restores Boltzmann equilibrium FT 0 0 • T2-relaxation (transverse relaxation)spin-spin) 0 50 75 100 125 0 5 10 15 20 25 30 35 40 25 150 175 200 freq. (s -1 ) time (ms) – disappearance of transverse (x,y) magnetization – contributions from spin-spin and T1 relaxation – 1/T2 ~ signal line-width • T1-relaxation (longitudinal relaxation / spin- FT lattice) – build-up of longitudinal (z) magnetization – determines how long you should wait for the next experiment 31 32 Relaxation Relaxation • Restoring Boltzmann equilibrium • Restoring Boltzmann equilibrium • T 2 relaxation: disappearance of transverse (x,y) • T 1 relaxation: build-up of longitudinal (z) magnetization magnetization !! T 1 determines when to start the next !! 1/T 2 ~ signal line-width !! experiment !!

33 34 NMR spectral quality Scalar coupling / J-coupling • Sensitivity – Signal to noise ratio (S/N) • Sample concentration • Field strength H 3 C - CH 2 - Br • .. • Resolution 3 J HH – Peak separation • Line-width (T2) • Field strength • .. 35 Key concepts NMR • Nuclear magnetic resonance • In a magnetic field magnetic nuclei will resonate with a specific frequency • FT-NMR Multidimensional NMR • Pulse, rotating frame, FID • Chemical shift • Electronic environment influences local magnetic field -> frequency • NMR relaxation • T 1 & T 2 • J-coupling

38 Why multidimensional NMR? 2D NMR • Resolve overlapping signals • observe signals from different nuclei separately • Correlate chemical shifts of different nuclei • needed for assignment of the chemical shifts • Encoding structural and/or dynamical information • enables structure determination • enables study of dynamics 39 40 3D NMR nD experiment direct dimension 1D 1 FID of N points t 1 preparation acquisition indirect dimensions 2D t 1 N FIDs of N points t 2 preparation evolution mixing acquisition 3D NxN FIDs of N points t 1 t 2 t 3 preparation evolution mixing evolution mixing acquisition

41 42 Magnetization transfer Encoding information • mixing/magnetization transfer • Magnetic dipole interaction (NOE) dipole-dipole interaction – Nuclear Overhauser Effect – through space – distance dependent (1/r6) ???? E = E = – NOESY -> distance restraints • J-coupling interaction – through 3-4 bonds max. proton A proton B – chemical connectivities – assignment spin-spin interactions – also conformation dependent 43 2D NOESY homonuclear NMR • Uses dipolar interaction (NOE) to transfer NOESY magnetic dipole t 1 t m t 2 magnetization between protons interaction crosspeak intensity ~1/r 6 – cross-peak intensity ~ 1/r 6 FID up to 5 Å – distances (r) < 5Å COSY t 1 t 2 J-coupling interaction transfer over one J-coupling, i.e. max. 3-4 bonds FID diagonal H N H N TOCSY J-coupling interaction t 1 t 2 transfer over several J- cross-peak mlev couplings, i.e. multiple steps FID over max. 3-4 bonds 44

45 46 Homonuclear scalar coupling 2D COSY & TOCSY 2D COSY 2D TOCSY H β H β H α H α 3 J H α H β ~ 3-12 Hz H N H N 3 J HNH α ~ 2-10 Hz 47 48 heteronuclear NMR homonuclear NMR ~Å E = E = NOESY proton B t 2 proton A t 1 t m E = E = FID A ( ω A ) A ( ω A ) (F1,F2) = ω A, ω A A A B ( ω B ) (F1,F2) = ω A, ω B B 1 H 15 N ω A ω B – measure frequencies of different nuclei; e.g. 1 H, 15 N, 13 C – no diagonal peaks Diagonal – mixing not possible using NOE, only via J F1 ω A Cross-peak F2

49 50 15 N HSQC J coupling constants – Backbone HN – Side-chain NH and NH 2 1 J CbCg = 35 Hz 1 J CbHb = 130 Hz 1 J CaCb = 35 Hz 1 J CaC’ = 1 J NC’ = 1 J CaN = 55 Hz -15 Hz -11 Hz 1 J CaHa = 140 Hz 1 J HN = -92 Hz 2 J CaN = 7 Hz 2 J NC’ < 1 Hz 51 52 1 H- 15 N HSQC: ‘ protein fingerprint ’ 1 H- 15 N HSQC: ‘ protein fingerprint ’

53 Key concepts multidimensional NMR • Resolve overlapping signals • Mixing/magnetization transfer Relaxation & dynamics • NOESY, TOCSY, COSY • HSQC • 3D NOESY-HSQC, 3D TOCSY-HSQC • Triple resonance 
 55 56 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 B 0 relaxation B 1 • Return to z-axis – Spin-spin relaxation – Transversal relaxation → T2 B 0 relaxation B 0 • Dephasing of magnetization in the x/y B 1 plane + return to z-axis B loc