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Biochemical Applications of Nuclear Magnetic Resonance

by: Dr. Pablo Pollich

Biochemical Applications of Nuclear Magnetic Resonance BIOCHEM 801

Marketplace > University of Wisconsin - Madison > Biochemistry > BIOCHEM 801 > Biochemical Applications of Nuclear Magnetic Resonance
Dr. Pablo Pollich
GPA 3.75


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Class Notes
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This 40 page Class Notes was uploaded by Dr. Pablo Pollich on Thursday September 17, 2015. The Class Notes belongs to BIOCHEM 801 at University of Wisconsin - Madison taught by Staff in Fall. Since its upload, it has received 54 views. For similar materials see /class/205194/biochem-801-university-of-wisconsin-madison in Biochemistry at University of Wisconsin - Madison.


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Date Created: 09/17/15
Chemical shifts and coupling W Milo Westler Jan272009 Bchm 801 Unique chemical shifts for different atoms Peak intensity proportional to relative number of nuclei Jcoupling interactions between nuclei Dynamics linewidth of OH peak Ethanol Nuclear Magnetic Resonance NMR spectroscopy Relies on the magnetic moments present in certain atomic nuclei The nuclei most often studied in biological applications of NMR are 1H 13C 15N and 31P Natural abundances 1H 100 13quotC1 15N04 and 31P100 These all have spin 1l2 which makes them more friendly All of these nuclei are stable nonradioactive isotopes There NMR active nuclei that are radioactive such as 3H NMR spectroscopy When a molecule is placed in a magnetic field the nuclei act like tiny bar magnets and align like iron filings in the external magnetic field The nuclear bar magnets can align either in the same direction up or in the opposite direction down as external magnetic field these two alignments have different energies An analogy of this energy difference can be physically experienced by holding the north poles of two magnets together versus holding the north and south poles together In the first case they repel and in the latter they attract It is the difference in energy between these two alignments of the nuclei in the magnetic field that is the fundamental quantity that is measured by NMR NMR spectroscopy The great strength of NMR comes from the fact that the energy difference between the up and down states measured as the chemical shift is exquisitely sensitive to the molecular environment of the nucleus Virtually every nucleus in a molecule experiences a different environment and thus has a different chemical shIft from every other nucleus In the molecule The structure of a molecule is encoded in the chemical shifts of the nuclei Using rather sophisticated experiments the chemical shift of every NMR active nucleus in the molecule and its physical location in the molecule can be determined A number of other useful magnetic interactions among the nuclei are also measured by NMR couplings Nuclear Spin Quantum effect Theoretically obtained from relativistic Dirac equation Classical analog to bar magnet magnetic dipole Spin 12 S12 Two energy levels on amp B Spherically shaped nuclei Relaxation mechanisms all relatively weak interactions Spin S1 or greater Lu spin 7 ZS1 energy levels Nonspherical shape of nucleus gives rise to quadrupole moment Strong relaxation pathway due to interaction with electric field gradients at the nucleus Not very useful for large molecules broad lines NUCLEAR SPIN DDDDDDDDDBEDD Dmmmmmmmmam Energy due to nuclear spin in a magnetic field E th01m 9 magnetogyric ratio h Planck s constant H0 magnetic field strength lm i12 on or 5 spin state Boltzmann Equilibrium N 3 2 e AE cT N05 NX is the number of spins in the x spin state AE is the energy difference between the on and B states k is the Boltzmann constant T is the absolute temperature For a 1409 T 600 MHz1H magnetic field the AE is roughly 60 mcallmole At room temperature 600 calmole thermal energy this corresponds to an excess of about 1 part in 10000 more on than B spins By comparison in optical spectroscopy the AE is roughly 6000 calmole blue light and the population of the first excited energy level is about 1 part in 20000 BL a1 B00 Place in magne c field 000000 B0gt0 Inmalsfo re 4 Relaxation o o o Ne r magne c moment a1 TY X V3 m1 hquot gNBNH Energy l 0 H Field FIG 11 Proton spin levels in a magnetic eld Becker High resolution NMR Academic Press New York 1969 Nuclear magnetic resonance frequencies IMHz Nucleus 1174T 14097 1644T 1879T 2114T spin 1H 12 5000 6000 7000 8000 9000 19F12 4704 5645 6585 7526 8467 31P 12 2024 2429 2834 3238 3643 13C 12 1257 1509 1760 2012 2263 15N12 507 608 709 811 912 2H 1 768 921 1075 1228 1382 Multinuclear NMR I5N 2H 13C 31F 19F 39IH I I I I I 100 200 300 400 500 MHz Why higher magnetic field Sucrose 60 MHz f 6 up no 3f V in n H H0 7 a 0 l ltcuzorl 47 ppm HDO T3139 H 6M4 H RGS IIADlt ism rL H InM Sucrose 500 MHz a Why higher dimensions Tsp 10mM Cc2 rm isp OmM C One benefits of Jcoupling H i i 39i39 39F if 4 20 0707 H H H 5NHSQC o HCH 2Dimensional NMR N ppm chemical shift H ppm chemical shift Black no Ca2 Red 10 mM Ca2 o av39 I O n O 1 u 39 W 5 39 2Dimensional 7 511 mnz HNCO i M HC IH H O glee ll IL H I O HCH 3 dimensional NMR 3Dimensional H 5NHSQC we 0 56 0 s amp 0 n r o 9 0 3939 59v maz HNCO 1 C175ppm o 0amp3 was 0 9 D m 1 55a maz I 98sz a an 93 Izogm 3 maam 0 16092 1 was 9 9960 5 In P 1 mxzoovz 2 3 NMR is not limited to 3 dimensions 45 D Reduced dimensionality experiments 3D gt 2D 4D gt 3D MD gt ND Bottom line More resolution and more connectivities Chemical shifts arise from electron currents in molecules BiotSavart Law mum quotoldLN i 3 4 M in r2 In dl mum m Inquu nr annular mm Ilium ml I 12 Illquot vulnr In Imam llmllnn n I m valor mm quotmm III cm In In mld pnInI Chemical shielding H M 10 20 30 C Will I Fl ll l lllll N i ll ll l ll l l I 400 200 0 200 400 chemical shielding ppm Inductive effects diamagnetic m oa ins m 42 oz 04 ova as m pw r 61 mm p m a c I H HN H 39 VF 1 H H H HNH o M HD on 1 1 Hl Hm D Hp O ow 0u 039 01440 MP H 4 H L H le lt I l H H c H MM H H M W 0 99 O OH O 339 Chick quot043 1 ppm Isotropic and anisotropic magnetic susceptibility Anisotropic Isotropic Ring currents 0 50 020 3 O 39 0 100 2 5 200 20 r 300 Z 400 15 10 g i 05 ii i 200 450100080 O60 040 39020 O10 r w I l l l l l I l l 1 O 05 10 15 20 25 30 35 40 45 50 p Fig 113 The shielding zone about a rapidly tumbling benzene ring The plot represents one quadrant of a plane through the center of the ring and normal to the ring plane The origin of the plot is at the ring center and the p axis is in the ring plane p and z are in units of 139 A The isoshielding lines are in ppm where positive numbers indicate shifts to higher eld From Johnson and Bovey Becker High resolution NMR Academic Press New York i 969 Tm i a b FIG 33 a This shows the induced diamagnetic circulation of nelectrons in a double bond for one of the orientations between bond and eld b The positive and negative shielding regions are shown as the average effect for all mutual orientations The pictures apply approximately also for gtC0 gt028 gtCN The intensity of the effects in the legions diminished outwards from the multiple bond and radially within each region The deshielding regions are ellipsoidal cones of aperture 114 and 104 in the X Y and XZ planes respectively Becker High resolution NM R Academic Press New York1969 Chemical exchange Bain 2001 N MR Time Scale Time Stale Chem Slmftj Cunp ng bust J T2 relaxation Slow 1i lt31 EA 513 k lt31 JV JB Intemlediate k EiA SB 1 JA TB Ara5t k lei EA GB quotbi e JA IE See391 U 10 G 0 12 k tici T3 T2513 1 Thi T1213 k 1 Im TTB 1 20 39 NMR timescale refers to the chemical shi timescale The range of the rate can be studied DDS5000 5391 far H can be extended to faster rate using 19F 13C and etc B Bound llgand M Free 111lnbxtor c magi ugmm7U A U ml Wi i312 JQ W Lam xhqt 1 I J 12 M f j W 11 Sim 1M 10 S M m m m my V my Spinspin coupling Nuclearnuclear coupling Fine structure in NMR spectra J scalar coupHng Main player in multidimensional NMR Dipolar coupling averages to zero in isotropic solution Affects relaxation Electronnuclear coupling hyperfine structure in ESR hyperfine shifts in NMR Fermi contact Pseudocontact dipolar Electronelectron coupling fine structure in ESR spectra CH3 CH2 GRO FREQ DIFF4 J 65 HZ UP D 5 PPM it 1 SPIN SIMULATION AT 10 MHZ AT 60 MHZ AT 200 MHZ AT 300 MHZ F m o 1 ma 1 4 39 1 L13 L 00 I 50 I12 Indirect nuclearnuclear spinspin coupli I 98sz a an 93 Izogm 3 maam 0 16092 1 was 9 9960 5 In P 1 mxzoovz 2 3 J HNAHu Cauphng Constant Hz a 3n Tarsmn Ang e 0 Torsmn 3779 s 50 an 120 15D 151 529 0 u HN 7 HM Couphng H Consmm 00 co Newman memoquot nf me C N Band cf a Pratem Calibration of J coupling constants 3JHNH Hz Figure 4 Measured values for 94 3JIINH coupling constants obtained from the HNHA spectrum of the protein SNase plotted as a function of backbone angle o 3JH IIquot couplings to the H 3 of glycine residues are indicated by 4 The backbone angle o is offset by 120 for these data points Lys70 and Lys78 are marked by squares see text The solid line indicates the leastsquares minimized t of the Karplns curve eq 5 with A 651 B 176 and C 160 and a rmsd of 073 J Acos2 60 Bcos 60 C Vuister and Bax J Am Chem Soc Vol 115 No 17 1993


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