1. 1 Modern Approaches to Protein structure Determination (6 lectures) Dr Matthew Crump

2. 2 Lecture Plan 1. Introduction to NMR (a refresher) 2. Solving Protein Structures by NMR - The features of a 1D spectrum - what can we tell? The need for 2D 3. 2D NMR - How NMR works through space not just bonds - we need this to solve structures. The move to the third dimension 4-5. Modern methods for structure determination 6. Comparison of techniques and New developments

3. 3 Lecture 1. Introduction to NMR. NMR = Nuclear Magnetic Resonance NMR is the most versatile tool in the physical sciences. May be used to probe the large-scale structure of objects -- including the anatomy of the human body May be used to probe the small-scale structure of molecules -- including the 3D structures of proteins and nucleic acids May be used to detect motion on a macroscopic scale (examples: flow, diffusion) and on a microscopic scale (example: molecular flexibility) knee spine DNA Duplex Prion Protein

4. 4 History of nuclear magnetic resonance 1946 Bloch, Purcell First nuclear magnetic resonance 1955 Solomon NOE (nuclear Overhauser effect) 1966 Ernst, Anderson Fourier transform NMR 1975 Jeener, Ernst Two-dimensional NMR 1985 Wüthrich First solution structure of a small protein NMR is about 25 years younger than X-ray crystallography 1987/8 3D NMR + 13C, 15N isotope labeling 1996/7 New long-range structural parameters: residual dipolar couplings (also: anisotropic diffusion 2003First solid state NMR structure of a small protein Nobel Prizes 1944 PhysicsRabi (Columbia) 1952 PhysicsBloch (Stanford), Purcell (Harvard) 1991 ChemistryErnst (ETH) 2002 ChemistryWuthrich (ETH) 2003 MedicineLauterbur (Urbana), Mansfield (Nottingham)

5. 5 Why study protein structure? The more we understand about a protein and its function, the more we can do with it. It can be used for a new specific purpose or even be redesigned too carry out new useful functions (biotechnology & industry). We can use this knowledge to help understand the basis of diseases and to design new drugs (medicine & drug design). The more knowledge we have how proteins behave in general, the more we can apply it to others (protein families etc) Complex, could be the active form

6. 6 Why biomolecular NMR? Structure determination of biomacromolecules -no crystal needed, native like conditions - nucleic acids: difficult to crystallize, affected by crystal packing Ligand binding and molecular interactions in solution. - Bandshift” in NMR fingerprint - with residue/amino acid resolution !!! Characterization of dynamics and mobility (ps to s) -conformational dynamics. enzyme turnover, kinetics, folding Molecular weight: X-ray > 200 kDa, NMR < 50-100 kDa - NMR and X-ray crystallography are complementary

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8. 8 Felix Bloch Edward Purcell

9. 9 Apply radio frequency to measure the nuclear precession frequencies at

10. 10 Two types of angular momentum “Normal” or “extrinsic” angular momentum (due to rotational or orbital motion) “Intrinsic” or “spin angular momentum” (a property of fundamental particles -- cannot be visualized). use your right hand to figure out the way the angular momentum vector points the direction of the spin angular momentum is indicated by an arrow.

11. 11 Quantum Angular Momentum If we specify an I value, quantum mechanics restricts us as well to specifying the projection of this vector along only one of the three Cartesian components of I. By convention the z-axis is chosen and I z is given by where m is a second quantum number which can take values m=-I,-I+1,-I+2,..,I. Therefore I z has 2I+1 values. In quantum mechanics, angular momentum is quantized. The total angular momentum of particles with spin takes the values of the form

12. 12 The gyromagnetic ratio  determines the ratio of the nuclear magnetic moment to the nuclear spin. It is a fundamental property of each nuclear isotope Fundamental symmetry theorems predict that spin and magnetic moment are co-linear Gyromagnetic ratio (1) The gyromagnetic ratio is also known as the magnetogyric ratio   =  I This equation tells us how much magnetism we get for a given spin.

13. 13 Nuclear Spin The magnetic dipole moment  and therefore the precession frequency are characteristic for each Nucleus and scale with the gyromagnetic ratio. Atomic nuclei are composed of protons and neutrons which have a spin Protons spin neutrons spinnuclear spinm Eveneven0 0 Evenodd1/2<0 Oddeven 1/2 >0 Oddoddn>0 NMR properties of selected nuclei NucleusI  s) -1 rad  rel Natural Abundance (%) 1 H1/22.6752 x 10 8 1.00 99.98 2 H14.107 x 10 7 0.150.02 13 C1/26.728 x 10 7 0.251.11 14 N11.934 x 10 7 99.64 15 N1/2-2.712 x 10 7 0.10.36 17 O5/2-3.628 x 10 7 0.04 19 F1/22.5181x10 7 100 23 Na3/27.080 x 10 7 100 31 P1/21.0841 x 10 8 0.41100 113 Cd1/25.934 x 10 7 12.26

14. 14 the energy of the state with quantum number I z is given by Zeeman splitting Planck constant gyromagnetic ratio Energy of interaction is given by E=- .B in a magnetic field B. The dot product tells us the energy depends on the size and relative orientation of B and . We take B o to be along the Z axis, so the dot product becomes E=-  z B z(o) (i.e.  x B z and  y B z = 0)

15. 15 m=-1/2 m=+1/2 I=1/2 m=-1 m=+1 I=1 m= 0 The Zeeman splitting is therefore ground state; no field ground state; with field Zeeman splitting Energy

16. 16 rad s -1 rad s -1 T -1. T s -1 (Hz) Larmor Frequency

17. 17 A compass in a magnetic field

18. 18 A nuclear spin precesses in a magnetic field the circulating motion of the spin angular momentum is called precession Nuclear spins precess because: they are magnetic they have angular momentum this arrow denotes the direction of the spin angular momentum

19. 19 Precession frequency = Larmor frequency 0 = -  B o /2π Larmor frequency in Hz (= cycles per second) gyromagnetic ratio in rad s – 1 T –1 magnetic field in Tesla (T) Compare with Zeeman Splitting

20. 20 Zeeman Levels

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22. 22 NMR signal Note the orientation of the coil -perpendicular to the magnetic field x y z BoBo Preamp The NMR signal is also called the free induction decay (fid)

23. 23 Fourier transformation and the NMR spectrum Fourier transform The Fourier transform (FT) is a computational method for analyzing the frequencies present in an oscillating signal The NMR spectrum RF Pulse

24. 24 A one-dimensional (1D) NMR spectrum of a protein HNHN HH Chemical shifts in parts per million (ppm) Are independent of the field strength of the Static magnetic Bo field. See the supplementary lecture material and Rattle, ‘NMR Primer for Life Scientists Pages 19-21, 26. 9 Backbone H N Aromatics H2OH2O HH Upfield shifted methyls Methyl 8 7 65 4 3 2 1 0 1 H chemical shift (ppm) 600134800 600132400600130000 (Hz) 600.130000 (MHz)

25. 25 Despite all this 1D NMR can provide some information 1.) As we saw we can study the ‘global’ appearance of the 1D spectrum. 2.) It can be very specific - we can monitor just one clearly resolved signal - even if it is in a large protein. 3.) It works in solution - can monitor the effects of temperature, pH, buffer conditions and stability over time.

26. 26 Y215Q

27. 27 http://www.chm.bris.ac.uk/polyketide/nmr.htm http://www.chm.bris.ac.uk/polyketide/nmr.htm Will have Lecture 1 (overheads) Plus Notes on Basic NMR.