Nuclear Magnetic Resonance (NMR) Spectroscopy Felix Bloch & Edward Purcell Richard Ernst Kurt Wuthrich Physics-1952 Chemistry -1991 Chemistry-2002 Paul Lauterbur & Peter Mansfield Brian Sykes Lewis Kay Medicine-2003 Principles of NMR Protein Spectroscopy What does NMR tell us ? 1) Primary structure characterization 2) Dynamics - psec-sec timescales 3) Equilibrium binding 4) Folding/Unfolding 5) Three dimensional structure Some Advantages 1) Solution based 2) Non-destructive 3) Residue specific information Principles of NMR Protein Spectroscopy Wavelength (nm) X-rays Radio waves 10 100 1000 104 105 106 107 108 109 Crystallography IR NMR UV/Vis Electron Nuclear spin transitions transitions Principles of NMR Protein Spectroscopy Nucleus I # Protons # Neutrons 1H 1/2 1 0 12C 0 6 6 13C 1/2 6 7 14N 1 7 7 15N 1/2 7 8 Absorption of energy by nucleus - depends on nuclear spin (I) = sum of unpaired protons + neutrons (spin 1/2) I≠0 - NMR observed - spin will have magnetic moment µ=γI For proteins 1H, 13C, 15N (31P) Principles of NMR Protein Spectroscopy N N S Bo S N m = -1/2 mI = +1/2 I α β S m=(-I, -I+1 ….I-1, I) Principles of NMR Protein Spectroscopy Normal Magnetic Field (Bo) z µ=-γIz x y Bo Principles of Protein NMR Spectroscopy So how does this give us an “NMR signal” ? N N S β S N m = +1/2 m = -1/2 I I α S α β Principles of Protein NMR Spectroscopy This occurs for all 1H, 13C, 15N in magnetic field β γ (∗107 rad / T sec) 1H 26.75 13C 6.73 α 15N -2.72 B0 Principles of Protein NMR Spectroscopy β ΔE = γhB0 = hν ν = γB0 (Hz) ω = γB0 (rad) 2π α Larmor Precession Nucleus γ (∗107 rad / T sec) ν at 14.09 T 1H 26.75 600.00 13C 6.73 150.87 15N -2.71 60.82 Principles of Protein NMR Spectroscopy NMR is an insensitive method 1H N=106 11.75T 18.79T β 499,980 499,968 α 500,020 500,032 Principles of Protein NMR Spectroscopy Sensitivity nα-nβ = Δn = NγhBo 2kT 2 Mo=nαµzα + nβµzβ = Δnµzα= 1 γhΔn = 1 N (γh) Bo 2 4kT z z x x y y Bo Bo Principles of Protein NMR Spectroscopy Net Magnetization z z Mo x x y y ωo Bo Bo Rotating Frame Principles of Protein NMR Spectroscopy How is the NMR Signal Obtained ? -employ a rf pulse at ν of desired nucleus z z θ Mo x “rf pulse” x Bo y y B1 B1 Principles of Protein NMR Spectroscopy z z Mo π/2 = /2 x θ π x Bo y y B1 B1 pw = 6 µsec γB1 = 41 kHz rf on off Principles of Protein NMR Spectroscopy z z z Mo π/2 x x x Bo y y receiver y B1 B1 B1 rf FT on off time ν Principles of Protein NMR Spectroscopy z -t/T Mz(t) = Mo(1-e 1) z x T1 y B1 x z y receiver B1 T2 x y -t/T off B1 My(t) = My(0)*e 2 time Principles of Protein NMR Spectroscopy T1, T2 (sec) Linewidths 100 T1 10 1 0.1 T 0.01 2 0.001 0.5 Hz 10 Hz 100 10 1 0.1 0.01 0.001 Correlation Time, τc (nsec) MW 100 MW 20,000 Molecular Weight Principles of Protein NMR Spectroscopy NMR Instrumentation Magnet - Bo 18.79 T vacuum N2(l) He(l) magnet 22.31 T (2006) probe Principles of Protein NMR Spectroscopy Magnet Technology Principles of Protein NMR Spectroscopy Magnet Technology Principles of Protein NMR Spectroscopy Probe Technology B1 Bo Principles of Protein NMR Spectroscopy Cold “Cryogenic” Probe 1/2 S/N ~ 1/{Rs*(Ts+Tpa)+(Rc*(Tc +Tpa)} Tpa, Tc - lowered 298˚K 20˚K Rs, Ts - near 298˚K 3-4 time more sensitive 500 MHz + cold probe = 1.6x S/N 800 MHz Principles of Protein NMR Spectroscopy Block Diagram of NMR Spectrometer Probe obs, dec, lk obs, lk Duplexer Transmitter Preamplifier obs, lk CPU Receiver Computer.