NUCLEAR MAGNETIC RESONANCE : ADVANCES IN CHEMISTRY AND BIOLOGY Ryszard Stolarski

1. NUCLEAR MAGNETIC RESONANCE: ADVANCES IN CHEMISTRY AND BIOLOGY Ryszard Stolarski Division of Biophysics Institute of Experimental Physics Faculty of Physics Warsaw University

2. NMR in biophysics, chemistry, biology and medicine • Stern-Gerlach experiment (1921) • Resonance method and determinations of nuclear moments by • atomic- and molecular - beam experiments (1938) • Detection of nuclear magnetic resonance in bulk matter (1946) • Nobel prize in physics for F. Bloch (Stanford) and E. M. Purcell • (MIT) in 1952 r. • Fourier transformation (1960/1970) and two-dimensional NMR • of small proteins and nucleic acid fragments (1970/80) • Nobel prize in chemistry for R. Ernst (ETH, Zurich) in 1991 • Nobel prize in chemistry for K. Wüthrich (ETH, Zurich) in 2002 • Nuclear magnetic resonance imaging (MRI) of intact bodies • Nobel prize inphysiology and medicine for P.C Lauterbur • (Stony Brook) and P. Mansfield (Nottingham Univ.) in 2003 • Multidimensional (3D and 4D NMR) in structural determinations • of proteins and nucleic acids up to ~40 kDa (1990/2000) • TROSY and CRINEPT experiments; spectrometers with magnets • over 18 T (800 MHz); cryoprobes (2000 - )

3. High resolution NMR Liouville-von Neumann equation  - density matrix R - relaxation superoperator (relaxation times T1 i T2) interaction with the exciting field B1 chemical shifts j scalar couplings Transformation of the density matrix after B1 pulse of tp duration s = x, y Observables: x- and y-components of magnetization of N nuclei  in a molecule of concentration n in solution

4. Applications of NMR "Broad line" NMR: condensed matter investigations (biological membranes) Medical diagnostics: magnetic resonance imaging” (MRI) Studies of biochemical processes in intact cells: - in vivo NMR, - in cell NMR Studies of small molecules in solution: structure, dynamics, interactions, and physico- chemical properties Quantum computers Determination of structures and dynamics of biopolymers in solution

5. Biopolymers - nucleic acids and proteins Genome full set of genetic information in the body Proteome full set of proteins coded by the genes Genomics sequencing of DNA and identification of the genes Proteomics complete characteristics of the proteome Transcription (mRNA synthesis) Translation (protein synthesis) mRNA Protein DNA nucleus cytoplasm Gene expression

6. Aims of structural proteomics • High throughput determination of structures of most proteins • coded by sequencedgenomes • Molecular mechanisms of interaction of proteins with ligands. • Sequence - structure - activityrelationship for groups of proteins • interacting in a metabolic pathway • Drug design; choice of suitable „targets” for chemotherapy

7. Structural parameters of proteins NMR RESONANCE ASSIGNMENT: chemical shifts j, scalar couplings J(i,j), and nuclear Overhauser effect (NOE) rij LOCAL Dihedral angles  • Scalar coupling constants J(i,j) Interproton distances rij • Proton NOEs Secondary structure (TALOS) • Chemical shifts j GLOBAL Mutual orientations of the molecular fragments • Residual dipolar couplings STRUCTURE

8. Protein structure in solution by multidimensional NMR 5 structures of BPTI (58 amino acids) by 2D NMR with the X-ray structure 20 structures of yeast eIF4E (217 amio acids) by 3D NMR  helix  sheet Wagner G. et al., J. Mol. Biol. (1987) 196, 611-639 Matsuo H. et al., Nature Struct. Biol. (1997) 4, 717-724

9. 1D NMR Single pulse experiment FID signal equilibrium detection pulse duration (s): pulse phase:  1D spectrum of BPTI Wüthrich K. et al., J. Mol. Biol. (1982) 155, 311-319

10. 2D NMR 1H,1H-NOESY pulse sequence Signal: 1H equilibrium evolution t1 mixing detection t2 2D spectrum of BPTI 1 (1H) 1 (1H) 2 (1H) 2 (1H) Wagner G & Wüthrich K, J. Mol. Biol. (1982) 155, 347-366

11. 3D NMR 3D protein spectrum and its 2D cross-section {15N/13C},1H,1H-HMQC-COSY 1H 15N or 13C equilibrium evolution t1 evolution t2 detection t3 constant  constant  Signal: Oschkinat H. et al., Angew. Chem. Int. Ed. Engl. (1994) 33, 277-293

12. Functional Magnetic Resonance Imaging (fMRI) MEDICINE "PHILOSOPHY" An fMRI Investigation of Emotional Engagement in Moral Judgement Joshua D. Greene, R. Brian Sommerville, Leigh E. Nystrom, John M. Darley, Jonathan D. Cohen Science (2001) 293,2105-2108 Magnetic resonance image of a transverse slice of a monkey head; contrast based on blood microcirculation Geoffrey Sobering, Science (1990) 250

13. Electric charge distribution in the 7-methylguanine ring of cap ? CH3 O H * N N O O O + H H H N O O O B O P P P CH2 CH2 H2N N O O H HO - - - H H O O O O H H H H O O C H 3 - P O O mRNA cap structure O

14. Examples of fitting the NMR signals using trial values of the couplings C2 GTP JC2-N2 = -23.4 JC2-N1 = -14.6 JC2-C4 = 7.8 JC2-N9 = -3.7 JC2-N3 = -3.0 C6 GTP JC6-C5 = 87.9 JC6-C4 = 12.9 JC6-C8 = 6.8 JC6-N7 = -7.9 JC6-N1 = -5.9 JC6-N9 = -1.3 C8 m7GTP JC8-N9 = -18.4 JC8-N7 = -18.4

15. Changes of the NMR parameters due to methylation at N7 of guanine: shielding constants () and reduced couplings (1K)

16. Changes of the calculated atomic charges due to methylation at N7 of guanine

17. Conclusion: localization of the net positive charge at N7 guanosine 7-methylguanosine

18. "Charge distribution in 7-methylguanine regarding cation- interaction with protein factor eIF4E" Biophysical Journal 85, 1450-1456, 2003 Division of Biophysics Katarzyna Ruszczyńska-Bartnik, PhD student Janusz Stępiński Edward Darżynkiewicz Institute of Organic Chemistry, PAS Krystyna Kamieńska-Trela Institute of Biochemistry and Biophysics, PAS Jacek Wójcik