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Published on October 15, 2007

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Biochem 925: NMR in Biochemistry:  Biochem 925: NMR in Biochemistry Guangshun Wang, Ph.D. Assistant Professor Director of NMR Core Facility The Structure-Fun Laboratory Eppley Institute for Research in Cancer and Allied Diseases, UNMC DRC6003, Biochemistry, Feb. 7, 2006 Outline:  Outline Some basics of NMR Peptide structure by 2D NMR Small protein structure by multidimensional and multinuclear NMR techniques NMR vs. x-ray NMR Tour Part I: Some basics of NMR:  Part I: Some basics of NMR An overview of NMR components and key structural parameters What is NMR? :  What is NMR? NMR: Nuclear Magnetic Resonance. This is an excellent name! Nuclear: You can do NMR on almost all elements as long as it has magnetic moment! (spin-half: odd atomic number (such as C13); even atomic and electron number: spin zero (NMR-inactive such as C12); even atomic number but odd electron number, spin > 1/2, such as H2). Magnetic: You need a magnetic field (Is there any exception?). Interaction between the magnetic field and the magnetic moment leads to the spitting in energy level. Resonance: You will see spin transitions between the energy levels in the presence of a radiofrequency field. Why bother with NMR?:  Why bother with NMR? Resolution! Resolution! Resolution! Site-specific structural information; The best spies in human bodies (purturbation-free, Non-invasive, extremely low energy ~ r.t.) Dynamics and function. The Nobel Prize in Physics 1952:  The Nobel Prize in Physics 1952 “ for their evelopment of new methods for nuclear magnetic precision measurements and discoveries in connection therewith ” Felix Bloch (1905-1983), Standford, USA Edward Mills Purcell (1912-1997), MIT/Harvard, USA Major components of a NMR spectrometer:  Major components of a NMR spectrometer Magnet; Probe; Console (synthesizer, transmitter, and receiver); User interface (SGI or Sun). A carton view of the NMR machine:  A carton view of the NMR machine 900 MHz NMR Magnets:  900 MHz NMR Magnets 900 MHz or 21 Tesla? :  900 MHz or 21 Tesla? The magnetic field B is constant (say 21.1 tesla); The gyromagnetic ratio is another constant (2.6752 x 108 T-1S-1); The resonance frequency can now be calculated using the NMR equation (in Hz) as below: o = B/2π = 2.6752 x 108 T-1S-1 x 21.1 T/(2 x 3.14159) = 898.38 MHz Proton is the most sensitive nucleus (cf. Gyromagnetic ratios of some other nuclei, 13C, 6.728 x 107; 15N, -2.712 x 107; 31P, ; 2H, 4.107 x 107). That’s why we say 900 MHz! The resonance frequency on a 900 MHz is 90 MHz for N15 and 225 MHz for C13 nuclei, respectively. The importance of chemical shifts:  The importance of chemical shifts If you had seen only one line in the ethanol spectrum, NMR would not have been so exciting! A period! As a consequence, the NMR equation need be modified to the following format:  = B(1-) Where  is the shielding tensor. Spin-Spin Coupling: the n+1 rule :  Spin-Spin Coupling: the n+1 rule Spin-Spin coupling: through bond:  Spin-Spin coupling: through bond 3JHX HN-H: J < 6 Hz in -helices; J > 8 Hz in -sheet; Definition of the dihedral angle  in the Karplus equation. J() = Acos2 + Bcos + c In principle, protein structures can be determined by measuring all dihedral angles. 1D NMR Spectra: from peptides to proteins:  1D NMR Spectra: from peptides to proteins The amide region (~6-12 ppm) and aliphatic region (-2-6 ppm) Top: 10mer; Middle: 46mer; Bottom: 154mer. The Nobel Prize in Chemistry 1991 :  The Nobel Prize in Chemistry 1991 Richard R. Ernst Eidgenössische Technische Hochschule Zürich (Swiss Federal Institute of Technology Zürich) Zürich, Switzerland b. 1933 "for his contributions to the development of the methodology of high resolution nuclear magnetic resonance (NMR) spectroscopy" From Continuous Wave (CW) to Fourier Transform (FT) NMR:  From Continuous Wave (CW) to Fourier Transform (FT) NMR Pulse Sequences for 1D and 2D NMR experiments:  Pulse Sequences for 1D and 2D NMR experiments In the 2D experiment, a pulse-delay unit has been added to encode the information in another time domain. In the 1D experiment, the frequency region of interest is excited simultaneously and FID collected afterwards. From 2D time domains to 2D frequency domains:  From 2D time domains to 2D frequency domains 2D Fourier transform NOE: through space :  NOE: through space NOE: nuclear Overhauser effect (Albert Overhauser, 1953, Phys. Rev. 91: 476). Perturbation of spin A will cause a change in intensity of spins in the vicinity. Since NOE is related to distance and molecular correlation time, it contains structural information. Indeed, this forms the basis for structural determination of biomolecules by NMR. Pulse sequences for NOE detection:  Pulse sequences for NOE detection 2D NOESY of lysozyme at 900 MHz Which is which?:  2D NOESY of lysozyme at 900 MHz Which is which? Protein Structure by NMR: The Wuthrich Approach :  Protein Structure by NMR: The Wuthrich Approach Distance restraints derived from nuclear Overhauser effect; Development of the 2D NMR techniques by R.R. Ernst (1974) based on Jeener’s idea (1971); Sequential signal assignment (1982); Conversion of the NMR restraints into a 3D structure using distance geometry (Crippen and Havel, 1970s). 2002: Kurt Wüthrich receiving his Nobel Prize from His Majesty the King at the Stockholm Concert Hall. :  2002: Kurt Wüthrich receiving his Nobel Prize from His Majesty the King at the Stockholm Concert Hall. Part II: Peptide structure by 2D NMR:  Part II: Peptide structure by 2D NMR The Wuthrich method 2D NMR Experiments for Synthetic Peptides:  2D NMR Experiments for Synthetic Peptides TOCSY (total correlated spectroscopy); DQF-COSY (double-quantum filtered correlated spectroscopy); NOESY (two-dimesnional nuclear Overhauser effect); HSQC (heteronuclear single-quantum coherence) Step I: Spin system identification by 2D TOCSY:  Step I: Spin system identification by 2D TOCSY NOE pattern for a helix (Wang et al., Biochemistry 1996; JBC 2005):  NOE pattern for a helix (Wang et al., Biochemistry 1996; JBC 2005) Protein Secondary structure: A proton network in protein with short distances observable by NMR:  Protein Secondary structure: A proton network in protein with short distances observable by NMR Alpha-helix: (I, I+1), (I, I+3), and (I, I+4) types of proton pairs within 5 Å. Beta-sheet: cross strand short distances. Between the secondary structures. Structure of a membrane-targeting sequence Wang et al. (2003) Protein Sci. 12: 1087.:  Structure of a membrane-targeting sequence Wang et al. (2003) Protein Sci. 12: 1087. Natural abundance HSQC and structural quality improvement (Wang et al. JBC, 2005):  Natural abundance HSQC and structural quality improvement (Wang et al. JBC, 2005) Part III: Structure of small proteins by nD NMR:  Part III: Structure of small proteins by nD NMR Pushing the molecular weight limit from ~10 kDa to ~100 kDa A typical path for NMR structure determination:  DNA cloning and protein expression A typical path for NMR structure determination Protein purification and concentration NMR spectra, 1D,2D,3D and 4D including NOE or other structural parameter measurements Squence specific signal assignments “Phase problem 3D structure generation based on NOE restraints 0.6 mL~1 mM protein solution ( 0.3 mL minimum) Secondary structure identification NOE pattern recognition Structure refinement and validation Protein Expression Systems :  Protein Expression Systems Escherichia coli Yeast Insects Plants Mice Cell free Protein/peptide purification followed by SDS-PAGE:  Protein/peptide purification followed by SDS-PAGE Stable isotope labeled samples:  Stable isotope labeled samples 15N; 15N,13C; 15N,13C,2H; 15N,13C,2H,Val,Ile,Leu methyl protonation. 3D NMR Spectra JACS 125, 13958 (2003):  3D NMR Spectra JACS 125, 13958 (2003) Spin-spin Coupling Constants in Proteins:  Spin-spin Coupling Constants in Proteins NMR pulse sequence for HSQC:  NMR pulse sequence for HSQC HSQC: Chemical shift mapping The principle of drug screening (Wang, Protein Sci. 2005):  HSQC: Chemical shift mapping The principle of drug screening (Wang, Protein Sci. 2005) A Set of Triple-Resonance Experiments:  A Set of Triple-Resonance Experiments A 3D HNCO Experiment:  A 3D HNCO Experiment Sequential signal assignments by triple-resonance NMR spectra: HNCO (black) & HN(CA)CO (gray):  Sequential signal assignments by triple-resonance NMR spectra: HNCO (black) & HN(CA)CO (gray) The way to NMR structures of proteins:  The way to NMR structures of proteins The first protein structure determined by NMR (JMB 1985). a. All heavy-atom presentation of the NMR structure of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA). b. Superposition of the core region of residues 23–42 in the NMR structure of BUSI IIA (green) with the corresponding polypeptide segment in the X-ray crystal structure of the homologous porcine pancreatic secretory trypsin inhibitor (PSTI) (blue). Part IV: NMR and X-ray diffraction are complementary to each other:  Part IV: NMR and X-ray diffraction are complementary to each other Why another technique for structural elucidation? :  Why another technique for structural elucidation? X-ray crystallography is the gold standard, but not all proteins can be crystallized; Sometimes the phase problem can not be solved for the crystal, or the crystal does not diffract well; Sometimes the structure determined in the crystal does not represent the form in aqueous solution; Even the crystal structure is available, NMR studies can offer additional insights such as dynamics; Joint structural refinement and cross validation. X-ray vs. NMR (Dimer in crystal (left) but monomer in solution (right) Wang et al. 2005 Protein Sci. 14:1082):  X-ray vs. NMR (Dimer in crystal (left) but monomer in solution (right) Wang et al. 2005 Protein Sci. 14:1082) Signal transduction Membrane proteins and protein complexes :  Signal transduction Membrane proteins and protein complexes Intermolecular NOE cross peaks Wang (2000) EMBO J 19, 5635.:  Intermolecular NOE cross peaks Wang (2000) EMBO J 19, 5635. Protein A and protein B are labeled differently. N15-C13 in this case. Signal transfer path is H1-N15-H1(A)-H1(B)-C13-H1, where NOE transfer occurs between HA and HB; Only signals between the two protein interface have the chance to survive. Three protein-protein complexes from the glucose pathway of E. coli Structure and function?:  Three protein-protein complexes from the glucose pathway of E. coli Structure and function? Phosphoryl transfer; Phosphorylation in regulation, for example, Ser46 of HPr (Wang, EMBO 19, 5635, 2000). The tail story (Wang, JBC 275, 39811, 2000; Protein Sci. 12, 1087, 2003). TROSY for Large Proteins Pervushin (1997) PNAS :  TROSY for Large Proteins Pervushin (1997) PNAS 1). TROSY: transverse relaxation optimized spectroscopy. 2). At high field, (DD + CSA) and (DD-CSA)! 3). The linewidth in 2D TROSY (a) is much narrower than that in the corresponding HSQC spectrum (b). Integral Membrane Proteins The structure of OmpX in DHPC micelles:  Integral Membrane Proteins The structure of OmpX in DHPC micelles E. coli outer membrane protein X (OmpX). Solution (micelles) and solid-state (lipid bilayers) NMR. X-ray (red) and NMR (green) For a review, see FEBS Lett. 555 (2003) 144-150. NMR of 723-residue malate synthase G by Lewis Kay’s lab:  NMR of 723-residue malate synthase G by Lewis Kay’s lab Important techniques: 4D NMR experiments; Partial deuteration; TROSY at high field (800-900 MHz); Residue-specific isotope labeling. Assignments: JACS 124, 10025-35 (2002); E. Coli protein fold (82 kDa): PNAS 2005. Order and Disorder: Heteronuclear NOE:  Order and Disorder: Heteronuclear NOE Structural Genomics Initiatives:  Structural Genomics Initiatives A many-lab collaborative effort on structures of water soluble free proteins by X-ray and NMR. There is also a team attempting to solve all membrane proteins from M. tuberculosis. What about Nebraska? In 2004, the Nebraska Center of Structural Biology was established. You’re welcome to go to The Journal Club to learn more! Further readings:  Further readings S.W. Homans (1992) A Dictionary of Concepts in NMR, Clarendon Press, Oxford; H. Friebolin (1988) One- and Two-dimensional NMR spectroscopy, VCH Publishes; K. Wuthrich (1986) NMR of Proteins and Nucleic Acids, Wiley & Sons. Frank J.M. van de Ven (1995) Multidimensional NMR in liquids, Wiley-VCH, NY. Slide57:  Phone: 94176 (office) E-mail:gwang@unmc.edu (specify Biochem925) Lab: Eppley Institute ECI3018

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