Nucle lear ar Mag agnetic ic Re Resonan ance – From Bas asic P Phys ysics to to Biomedical al Applicat ations Tai ai-huan ang Huan ang Inst. B Biomedical Sciences, A Academia Sinica April 12, 2016 (NTU/IAMS)
Ou Outl tlin ine 1. The Dawn of NMR – It is all Physics . 2. Exploiting the power of NMR – A party for all. Chemistry, biology, material science, and medicine. 3. Manipulation of nuclear spins - Spin gymnastics. 4. Biomedical applications – Work from our lab. - Packaging of SARS CoV nucleocapsid. - Mechanism of SUMO mediated signal transduction. - Macromolecular dynamics in solid and solution. 5. Look back on a wonderful journey.
1. Th The Dawn of of NM NMR – A A fertile gr grou ound for or physi sicist sts 1924 Pauli proposed the presence of nuclear magnetic moment to explain the presence of hyperfine shift in atomic spectra. 1930 Nuclear magnetic moment was detected using the refined Stern-Gerlach experiment by Estermann. 1939 Rabi et al first detected nuclear magnetic resonance by applying rf energy to a beam of hydrogen molecules. 1946 Purcell et al at Harvard reported nuclear magnetic absorption in parafilm wax. Bloch et al at Stanford reported nuclear magnetic resonance phenomenom in liquid water. 1940s-60s NMR theories were developed by physicists.
2. E Exp xploiting g the power of NMR – A par arty ty f for al all 1949 Chemical shift phenomenon was observed. 1960s - Ernst and Anderson intrlioduced Fourier Transform technique into NMR that increased NMR sensitivity by orders of magnitude. - Solid state NMR was revived due to efforts of Waugh at MIT. Application to material and polymer science insoluble proteins etc. - Biological application became possible due to the introduction of superconducting magnet and high power computers. - NMR imaging was demonstrated (Lauterbur at Stony Brook). 1970s - Development of multi-dimensional NMR (Jeneer, Ernest, Bax ..) - Development of methodologies for determining macromolecular structure (W ϋ thrich).
1980s and beyond – Exploding applications. - Methods for characterizing macromolecular structure/dynamics in solution matured. - Macromolecular structures in solid and gel states become feasible. - Material science: Zeolites, polymers, fuel cells etc. (Clare Grey in Cambridge on Li-Air battery 5x more compact) - MRI become a powerful clinical imaging modality. - Functional MRI come to stage. - Development of several fast NMR methodologies. - NMR-based Metabolomics. - …… Non-trivial applications. - Each become a sub-discipline by itself.
Nobel Laureates No s in NM NMR Isador I Rabi, Edward M. Purcell Felix Bloch Physics 1944 Physics, 1952 Physics, 1952 Peter Mansfield Paul C. Lauterbur Kurt W ὕ thrich Richard R. Ernst Physiol. Medicine, 2003 Physiol. Medicine, 2003 Chemistry, 2002 Chemistry, 1991
NM NMR Spectros oscop opy Radio Wave h n -1/2 Energy E = h n +1/2 B o B o = 0 B o Lar armor Equat ation ( (I = = ½ ½): n = B o / 2 n = Larmor frequency Bi Biologic ogical ally ly interested erested nucl uclei: ei: = nuclear gyric ratio 1 H, 13 13 C, C, 15 15 N, N, 19 19 F, F, 31 31 P (S=½), 2 D (S=1 =1) B o = magnetic field strength
Ba Basic Nuclear ar Spin In Inte tera racti tions 6 Electrons 3 3 2 1 1 H o Nuclear Spin i Nuclear Spin j H o 5 4 4 Phonons 7 Dominan ant t Inte terac acti tions: H = = H Z + H H CSA + H D + H H Q Q + H H J + … H z : Z Zeema man Int.; H CSA : Chemica cal Sh Shieldin ing g Anisot otrop ropic ic Int.; H D = : Dipolar ar Int. H Q : Q Quadrup upola olar r Int. H J : J-Coupli ling
Ba Basi sic Nu Nuclear Spin In Interaction ons Zeeman an Inte terac acti tion (H z .) (Field d depend) ; ; Interaction of nuclear spin with external magnetic field . H Q = = - γ I Z • Bo Chemical al Shielding Anisotr tropic ic Inte terac acti tion (H CSA ) (Field d dep.); The nuclear shielding effect of an applied magnetic field, caused by an The induced magnetic field resulting from circulation of surrounding electrons H CSA = = - γI • σ • B o Dipolar ar Inte terac acti tion (H D ) ( (Thru spac ace) ( (Field i indep): Interac actio ion n between en adjace cent nt nuclear r spins s through gh magne netic tic dipolar ar field.
Qu Quad adrupolar ar Inte terac acti tion (H Q ) : (Field indep) Nuclei with spin > 1/2 have a asymmetric distribution of nucleons (non spherical distribution of positive electric charge) H Q = I · V V · I I J-Couplings (Thru b bond connecti tion) : ( ( F Field indep) Resonance splitting mediated through chemical bonds connecting two spins. It is an indirect interaction between two nuclear spins which arises from hyperfine interactions between the nuclei and local electrons. 1 H 1 H 1 H
Magnitu itude de (Hz) Inte terac acti tion ( 1 H at 2.1T) 10 8 Zeeman 10 6 Quadrupole 10 3 Chemical shift 10 3 Dipole 10 J-Coupling The resonance frequency of a nuclear spin in single crystal depends on the orientation of the tensorial interaction w.r.t. the magnet field. Single crystal β 1 β 2
NM NMR sp spectrum of of sa samples s in so solid st states Po Powder pat atte terns
NMR spectra of samples in different states Small molecules in solution <H D > = <H Q > = 0 <H CSA > = σ iso ; <H J > = J iso Well-resolved sharp lines Macromolecules (Slow tumbling) Broad overlapping Gel state Gel state (Slow motion) (Featureless humps)
1. NMR spectra contains rich information derived from the presence of multiple interactions. 2. Each interaction provide insights into the structure/dynamics of the spin system. 3. It is difficult to quantify the interaction when there are more than one present. Question: How to extract the inter-twined interactions ? Design special pulse sequences to selectively observe/ suppress certain interaction(s) Spin gymnastics
Example: (HSQC) (2D Heteronuclear Single Quantum Correlation Spectroscopy) Fe Features: s: 1. Dramatically increased spectral resolution ! 2. Dramatically increased sensitivity of insensitive nuclei ! Enhancement factor ∝ (γ H / γ I ) 3 3. Opened a door for thru-bond sequential resonance assignment (Thru J-coupling). 4. The idea can be extended to higher dimension to include multiple nuclei and field gradients etc
NMR Spectroscopy Classical view Radio Wave h n Net magnetization Z RF field (B 1Y ) B o M = B 1Y τ M Z X B o M Y M X Y Magnetization will be flipped around Y-axis toward X-Y plane by an angle , determined by the RF field strength and the pulse duration. = 90 o it is call a 90 o pulse or /2 pulse (maximum signal) = 180 o it is call a 180 o pulse or pulse (No signal)
Pulse sequence for 15 N-HSQC expt Protein peptide chain Efficiency sin(2 J ); Maximum transfer when 2 J = /2.
RC-RNase (12 kDa) 15 N-HSQC of RC-RNase Each spot is a 1 H- 15 N pair of a residue Ser135 15 N 15 1 H
Bio iomedi dical al A App ppli licat atio ions Molecules Cell tissue Organ Whole body 1. Chemical Identification: A. Identification of metabolites (Metabonomics) B. Drug discovery. 2. Macromolecular structure: 2. Macromolecular structure: 3. Macromolecular Dynamics: 3. Macromolecular Dynamics: 4. Magnetic Resonance Imaging (MRI):
1. . Ch Chemic ical al I Ide dentif ific icat atio ion: Proton spectrum of ethyl acetate NMR MR spectr trum i is th the finger print o t of a c a chemical al Organic synthesis, natural product identification etc.
2. . Metab abonomic ics – (Nicholson an and L Lindon, Nat ature 455, 1 1054, 2 2008) Metabo bono nomi mics cs aims to measu sure re the global al, , dynamic mic metabol bolic ic respon onse se of living g system ems s to biologic gical al stimu muli li or genetic ic manipu pulat ation on. . It seeks s an analyt ytic ical al descrip iptio tion n of complex lex biologica gical l samples les and to charact cteri erize e and quantify ify all the small ll molecu cules les in such a sampl ple e (Urine, e, blood, plasma ma etc). ). Raw data (Urine, blood etc) Patt ttern recognition Statistical analysis Identi tify metabolites
NM NMR sp spectrum of of human u urine Ver ery y co comp mple lex x !
Pop opulation on st studies s sh show ow: Meta Me tabolic va variat ation is m much l lar arger th than an geneti tic va variat ation ! (Urina nary ry Metaboty otype pes) s) Japanese N = 1000 • Chinese N = 900 Americans N = 900
The World Phenome Center network
中研院台灣人體生物資料庫 (Taiwan Biobank) Collect and sequencing 300k samples (200K healthy, 100K patients of various diseases). (Already Collected over 60k samples now.) Perform genome sequence data of all samples for researchers performing other analyses (Data mining). Already identified diabetes markers from genome analysis. Hope to include NMR- and Mass-based metabonomics data.
2. . Mac acromole lecul ular ar structure/ e/fu funct ctio ion
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