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3. Nuclear Magnetic Resonance - NMR results from resonant absorption of electromagnetic energy by a nucleus (mostly protons) changing its spin orientation - The resonance frequency depends on the chemical environment of the nucleus giving a specific finger print of particular groups (NMR spectroscopy) - NMR is nondestructive and contact free - Modern variants of NMR provide 3D structural resolution of (not too large) proteins in solution - NMR tomography (Magnetic resonance imaging, MRI) is the most advanced and powerful imaging tool 347 Some history of NMR 1946 Principle of solid state NMR (Bloch, Purcell) 1950 Resonance frequency depends on chemical environment (Proctor, Yu) 1953 Overhauser effect 1956 First NMR spectra of protein (Ribonuclease) 1965 Fourier Transform spectroscopy (Ernst) 348 1973 Imaging tomography (Mansfield) 1985 First protein structure (bovine pancreatic trypsin inhibitor) in solution (Wüthrich) 349 By now: More than 150 protein structures (M < 60 000) BPTI Bound water Protein dynamics 350 Functional MRI 351 3.1 Principle of Nuclear Magnetic Resonance Many (but not all) nuclei have a spin (I). Quantum mechanically I can have 2I+1 orientations in an external magnetic field B. This spin is associated with a magnetic moment gI: nuclear g-factor 352 Since biomatter is made of H,C,N and O, these are the most relevant nuclei for biological NMR 353 Mechanical (classical) model Spinning top with magnetic moment mL and angular momentum I precesses with frequency wL under torque D B0 || z B1 Larmor precession of mL around B0 a y x Torque on magnetic moment mL in B0 Larmor precession around B1 The precession frequency is independent of a and equals the Larmor frequency Application of a horizontal magnetic field B1 which rotates at wL: In the frame rotating with mL the orientation of B1 relative to mL is constant Additional precession of mL around B1 at frequency 354 Quantum mechanical description The magnetic moment orients in a magnetic field B0. Different orientations correspond to different energies I = 1/2 1H, 13C, 31P gI = 5.58 B0 2H, 14N, E B0 g = 42.576 MHz/T I=1 mI = 1/2 mI = - 1/2 E B0 mI = 1 0 B0 -1 I = 3/2 E 23Na, B0 mI = 3/2 1/2 -1/2 When photons with frequency wL are absorbed a transition from the lower to the upper level occurs. Selection rule DmI = 1 B0 - 3/2 355 Bulk magnetization A sample contains many nuclei (typically N ~ 1017 or higher). In zero field all spin orientations are equivalent. The bulk magnetization (I.e. is the sum of all m’s) is very small and fluctuates around M=0. At finite fields B0 (and finite temperature) the occupation of states at different energies E obeys Boltzmann statistics exp(E/kBT) – thermal equilibrium is assumed. For I=1/2 the spin state “parallel” to B0 has lower energy E1 than the “ antiparallel” state with energy E2. Therefore there is a net magnetization along the z-axis. However since DE = E2 – E1 is much smaller than kBT the magnetization is far from saturation. 356 The number of spins in state 1,2 is Thus the population imbalance is Which yields a bulk magnetization with The average magnetization in x,y vanishes because the precessions of individual spins are uncorrelated. 357 The application of a pulse of duration t changes the average angle of the magnetization by a certain angle (c.f. the mechanical model or a change in population densities), given by: t g B1 Thus a pulse of duration t =2p/4 w1 gives a change in angle of p/2 – pulse I.e. the magnetization is flipped into the xy plane. Mx and My now oscillate with wL. If M is flipped out of equilibrium (out of the z-direction) by a B1- pulse, it will relax back to Mz into thermal equilibrium. This occurs because of magnetic interaction of m with the environment (atoms, eventually in crystalline lattice) and is characterized by the so–called longitudinal (or spin-lattice) 358 relaxation time T1. This relaxation is described by a set of rate equations for the transitions between the states dna W (n n0 ) W (na na0 ) dt dn W (na na0 ) W (n n0 ) dt Which yields a simple exponential relaxation of the magnetization in the z-direction 359 The amplitudes of Mx and My decay with another relaxation time T2 called spin-spin relaxation time. This relaxation originates from inhomogeneity of B0 . It is described by another phenomenological equation y y x x Immediately after p/2 pulse later 360 To be complete, the precession in the static field has to be taken into account as well, which is described by the Bloch equations One can detect the transverse magnetization Mx or My by a pick up coil where a current I(t) is induced by the oscillating transverse magnetization. The width of the FT of I(t) provides a measurement of T2 (Method of free induction decay) 361 3.2 Classical NMR experiments Absorption signal 362 600 MHz Proton NMR Spectrometer High frequency NMR spectrometers require very strong magnetic fields, which are produced using super-cooled coils (T = 4.2K, liquid He). The superconducting coils are surrounded by a giant vessel containing liquid N2. B0 He k N 2 B1 363 3.3 Chemical shift The external field B0 is changed (reduced in amplitude) due to local field -sB0 generated by the diamagnetic currents induced by B0 in the electron system near the nucleus. s is the shielding constant (diamagnetic susceptibility) The shielding depends on the orientation of B0 with respect to the molecules (e.g. benzene ring) near the nucleus. s is a tensor. If the rotational motion of the molecules is fast compared to 1/wL the precessing spin I sees an effective (time averaged ) field Bloc. If the rotation is free (like in most simple liquids) the anisotropy of the shielding is averaged out, s becomes a number. The NMR lines are very narrow. NB. In solids or large proteins in viscous environment where motions are strongly hindered or slowed down, the NMR lines are significantly broader. Motional narrowing! 13C NMR spectrum of liquid benzene 364 Usual measure: Frequency shift of sample (1) relative to some reference sample (2); unit: ppm Origin of chemical shift: = shielding of B0 365 Examples: 13C NMR Benzene C6H6 All 6 carbons are identical same chemical shift, one line Toluene C6H5-CH3 5 different types of C-atoms, 5 lines 366 1H-NMR of ethyl alcohol, CH3CH2OH Three types of protons CH3 OH CH2 368 Typical chemical shifts Reference Tetramethylsilane Si (CH3) 4 Has very narrow line Chemical shifts are frequently used in chemistry and biology to determine amount of specific groups in sample (quantitative 369 spectroscopy) 370 3.4 Pulsed NMR More efficient than classical (frequency or B) scans Study the free induction decay (FID) “Ideal” FID = one precession frequency Pick up coil 371 “Real” FID = several precession frequencies because of several nuclei with different chemical shifts 31P NMR FT 372 Spin echo Evolution = spreading (dephasing) in x,y plane 90 degree flip 180 degree flip = mirror image relative to x p/2 p Refocusing = spin echo My - echo after 2 t1 T1 T2 FID t1 t1 t 373 Spin-Spin Interactions give rise to relaxation of the magnetization Scalar or J – coupling (through bond) Most bonds are characterized by antiparallel orientation of electron spins (bonding orbital) The nuclear spins are oriented antiparallel to “ their “ bond electron eg H2 B A The nuclear spins mA and mB are coupled, independent of the direction of the external field; Interaction energy: DE = a mA . mB Energy to flip eg spin B A B NB: In polyatomic molecules the J-coupling can also be promoted by -Cbonds or other bonds ( A – C – B ). It is short ranged (max. 2 or 3 bond 374 lengths) J- coupling results in additional splitting of (chemically shifted) lines The magnetic dipoles of the CH3 group protons interact with the aldehyde proton spin and vice versa. Parallel orientations have higher energies. NB: the spin-spin coupling constant J also depends on the bond angle 375 -> info on conformation 1D NMR of macromolecules Alanine in D20 Lysozyme J-coupling (129 amino acids) Tryptophan in D20 J-coupling Assignment too complicated Assignment of lines ok structure NB: VERY high field NMR, in principle could 376 solve resolution problem Interactions between different spin-states Selection rule demands Dm 1 Gives rate equations of the type: dn1 Ws1 Dn2 Dn1 WI1 Dn3 Dn1 W2 Dn4 Dn1 dt 377 Generalizing from before, we obtain the magnetizations of the two spin states and the population difference: DI z Dn1 Dn3 Dn2 Dn4 DS z Dn1 Dn2 Dn3 Dn4 D2 I z S z Dn1 Dn3 Dn2 Dn4 Thus one obtains a rate equation for the magnetization: d DI z d Dn1 d Dn3 d Dn2 d Dn4 dt dt dt dt dt Which is more useful written in terms of magnetizations: d DI z WI1 WI 2 W2 W0 DI z W2 W0 DS z WI1 WI 2 2DI z S z dt Note selection rules demand W2 = W0 = 0 378 The same game can be played for the other magnetization, giving an analogue equation, which cross correlate the different spins. 2D NMR of macromolecules makes use of these cross correlations FID A second 90O pulse in the same (x) direction as the first one flips all spins pointing into y back to z. The instant Mx stays unaffected. Mxy Mxy(n) has marker at n1 = 1/t1 t t1 379 Protocol: Take FID’s at variable values of t1 1D (auto) peaks 380 Cross peaks indicating spin-spin coupling 2D COSY spectrum of isoleucine CdH3 CgH2 C H CaH Through bond interaction bewteen CaH and CH Cross peaks give information on distance along the bond 381 2D COSY spectrum of a heptapeptide Tyr-Glu-Arg-GlyAsp-Ser-Pro (YGRGDSP) 382 Direct dipole-dipole interaction (through space) can take up a change of Dm = +/- 1, I.e. relax the selection rules. B-field generated by dipole m Transition rates go with the square of the interaction VIS g 2 3 IS r , W0,2 g 4 rIS6 Related to the energy changes of A and B due to the induced fields at A and B: - mABB and - mBBA Strong dependence on distance between the different spin sites (r-6 due to dipole interaction) gives very sensitive spatial information about distances between spins down to 0.5 nm 383 Now take along the cross terms of the magnetizations gives the Solomon equation: R DIz I s DSz s DI z RS DS z Solved by: DI z t DI z t 0 exp Lt DS z t DS z t 0 RS RI 1 s RI RS 1 exp t exp t exp t exp t 1 2 1 2 2 2 R 2R 2R exp Lt s RI RS 1 RS RI 1 exp t exp t exp t exp t 1 2 1 2 2 2 2 R R 2 R 2 2 R R R 4s RI RS R 1 2 4s 2 1,2 I S 1 RI RS R 2 384 Simplify by assuming RI =RS: s 1 exp t exp t exp t exp t 1 2 1 2 2 R exp Lt s exp t exp t 1 exp t exp t 1 2 1 2 R 2 This implies maximum mixing after a time scale tm Flip the spins S at that time to enhance contrast 385 For macromolecules, there are many interacting spins, thus a much more complicated set of equations would have to be solved DI R1 s 1 j s i1 s n1 s nj s s in DI Rn 1n Combine this (Nuclear Overhauser) enhancement with the technique of 2D spectroscopy gives NOESY: The appearance of correlation peaks as a function of tmix gives information about the spatial properties (s) of the atoms 386 Part of 2D NOESY spectrum of a YGRGDSP H H NOESY correlates all protons near in real space even if the are chemically distant Typical NOESY signatures 387 Determination of protein structure from multi-dimensional NMR - data Starting structure (from chemical sequence) Random folding at start of simulation Heating to overcome local energy barriers Cooling under distance constraints from NMR Repeating for many starting structures Family of structures 388 389 NMR solution structures of proteins Tyrosine Phosphatase Cytochrome 3 390 3.5 MRI At much reduced spatial resolution, NMR can also be used as an imaging tool, where the spatial resolution is obtained by encoding space by a frequency (i.e. a field gradient) 391 Mostly driven by T2 relaxations, apply a gradient field across the sample, which gives different Larmor frequencies for different positions (all done at H frequencies) Resonance condition only fulfilled at one specific position 392 Now we have to also encode position in the x-y direction 393 Apply a field gradient along the y-direction for a short time, which gives a phase shift to the different nuclei as a function of depth 394 Finally apply a field gradient along the xdirection during readout, which gives a frequency shift of the FID precession 395 Then you take a signal with a pickup coil as a function of FID time and time duration of the phase coding pulse, which you Fourier transform to obtain a proper image 396 Since you have turned a spatial measurement into a spectroscopic one, the resolution is spectroscopically limited (or limited by the gradients you apply) Therefore fast scans (needed for functional studies have less resolution) 397 Recap Sec. 3 NMR is a spectroscopic method given by the absorption of em radiation by nuclei The signals depend on the nuclei, the applied field and the chemical environment Using Fourier-transform methods, a fast characterization of different freqeuncy spectra is possible Sensitivity is enhanced by using cross correlations in 2D NMR 398 More recap Dipole-Dipole interactions can be used to characterize spatial relationships Spin-Spin interactions are used to determine chemical bonds Gives atomic resolution for macromolecules including dynamics Using magnetic field gradients, spatially resolved measurements are possible resulting in MRI 399