Solid state and advanced NMR Dr. Magnus Wolf-Watz Department of Chemistry Umeå University magnus.wolf-watz@chem.umu.se NMR is useful for many things!!! Chemistry Structure of small molecules, chemical structure of catalysts Medicine MRI MRI of soft tissue like brain Functional MRI of metabolites, blood flow etc. Structure Biology High resolution NMR, structure determination of bio-molecules, dynamics NMR can be applied to liquid and solid samples 2
Six Nobel prices of the NMR phenomena 1943 (Physics) Otto Stern Discovered that the proton has a magnetic momentum. 1944 (Physics) Isodor Rabi Resonance method to record nuclear magnetic properties 1952 (Physics) Felix Bloch Measurements of a nuclear spin (the birth Edward Purcell of NMR) 1991 (Chemistry) Richard Ernst High resolution NMR and multidimensional NMR 2002 (Chemistry) Kurt Wütrich Bio NMR (protein NMR) 2003 (Medicine) Paul Lauterbur Development of MRI Peter Mansfield 3 The NMR experiment Prepare concentrated protein solution in a NMR tube Superconducting magnet Aquire data in time domain Fourier transform Transfor data To frequency domain Apply radio frequency radiation to exite spins. 4
Most commonly used nuclei in NMR Nuclei Isotope ratio (%) I (Spin quantum) Frequency (MHz) Comment 1 H 99.985 ½ 600 Most used 13 C 1.1 ½ 151 Bio & organic NMR 15 N 0.36 ½ 61 Bio NMR 19 F 100 ½ 564 Drug development 31 P 100 ½ 243 Nucleic acid membranes 27 Al 100 5/2 156 29 Si 4.7 ½ 119 17 O 4 5/2 81 5 Nuclear Angular moment (I) and the Magnetic Moment (μ) Most nuclei possess a nuclear angular moment, I. I can only have discrete values I is associated with the magnetic moment, μ. (μ = γi) The magnetic moment is the origin of the NMR signal. Hence nuclei with I=0 are NMR inactive (example 12 C, 16 O). 6
Nuclei in a static magnetic field If a nucleus with a nuclear angular moment, I, is placed in a static magnetic field it will orient so that I z = mħ m is the magnetic quantum number and can take the any of the values m = I, I-1, I-2,...,-I (2I+1) values of m. For a nuclei with I=½ the magnetic quantum number can be m= ½ and -½. For I = 1 the magnetic quantum number can be m= 1, 0, -1. I= ½ I= 1 7 Zeeman levels, for spin = 1/2 For instance hydrogen, 1 H, is a magnetic dipole, in the absence of an applied external magnetic field the orientation of these dipoles is random. Like a compass the dipoles can be aligned against or with the applied magnetic field when an external magnetic field is applied. Spins aligned with the magnetic field has lower energy. B o m=- ½ Net magnetic moment m=½ M z No magnetic field Applied magnetic field The net magnetic dipole moment is zero because all orientations are present and the resulting vector will be zero. The net magnetic dipole moment is a vector aligned with the aplied B O field.
The NMR signal Transitions between the different energy states are induced by a B 1 field (RF irradiation) with a frequency that mach ΔE, i.e. the resonance condition ΔE=γħB 0 =hυ 1 =has to be fulfilled. Frequency : υ=γ*b 0 /2 π When the B 1 field match the resonance condition the system is perturbed and magnetization in the xy-plane is created. E m=-½ (β) E β =+½γħB 0 ΔE=γħB 0 m=+½ (α) E α = ½γħB 0 9 B o M z B 0 x y RF y x M x Equilibrium, no signal. Only longitudinal magnetization RF pulse Rotating magnetization vector in the x,y-plane (transverse magnetization) produces a current in a detector coil
Detection of the NMR signal The NMR signal is detected when the magnetization is in the xyplane. This magnetization rotates in the xy-plane with a velocity corresponding to the chemical shift. This cause a current in a coil that is aligned along the x- or y-axis. 11 Free Induction Decay FID The induced current is cosine modulated and the corresponding signal represents the amplitude of the signal as a function of time. This is called Free Induction Decay, FID and represents intensity as a function of time. If there are more than one spin present the FID becomes very complicated to analyse. A Fourier transformation of the FID converts the FID to a spectrum, i.e. that represents intensity as a function of frequency. FT 12
The chemical shift The absolute frequency of a nuclei is dependent on the static magnetic field. υ=γ*b 0 /2 π Instead of frequency the chemical shift δ is used. ν δ = sample ν ν B o reference (MHz) reference 10 *106 6 (ppm) The chemical shift is independent of the static field strength. The chemical shift of a nucleus reports on the electronic environment. The ethanol spectrum contains information on purity and structure of the compound 13 Relaxation of the NMR signal After the excitation the system will go back to the equilibrium state. This is called relaxation, which is caused by small oscillating magnetic fields within the sample caused by thermal motion of the molecules. There are two types of relaxation processes, longitudinal relaxation, R 1, and transverse relaxation, R 2. R 1 re-establish the M z magnetization. R 2 describes how the signal disappears in the xy-plane, this rate is extremely important and can be used to study dynamics!!!! 14
M z = M o ( 1 - e -t/t1 ) M XY =M XYo e -t/t2 T 2 ~T 2 (Mw)+T 2 (Dynamics) Interactions intramolecular isotropic interactions chemical shifts J-couplings B 0 intermolecular anisotropic interactions chemical shift anisotropy Dipole-dipole coupling Quadrupole coupling = = not averaged out in solutions averaged out in solutions only relaxtion contribution 16
Chemical shift anisotropy, CSA In the presence of an external magnetic field electrons gives rize to a (small) local field at the nucleus. The nucleus exprienze the sum of the external and local field -> chemical shift. For most molecules the size of the local field (and hence chemical shift) depends on the orientation of the molecule with respect to the external field, i.e the chemical shift is anisotropic. In isotropic solution this effect is averaged to zero and we observe an isotropic chemical shift. In solids the chemical shift display an angular dependency of the form: ν(θ) = σ i + 1/3 Δσ (3cos 2 Θ-1) B 0 θ μ (isotropic chemical shift) 17 NMR-Resonance as a function of orientation, SOLID powder spectrum, CSA (chemical shift anisotropy) Static 31 P NMR 90 o B 0 0 o 54.7 o σ i Δσ ν(θ) = σ i + 1/3 Δσ (3cos 2 Θ-1)
Magic angle spinning in solid state NMR ν(θ) = σ i + 1/3 Δσ (3cos 2 Θ-1) Set the angle, β, to 54.7. ν(θ) = σ i + 1/3 Δσ (3cos 2 Θ-1) =0 ν(θ) = σ i (= isotropic chemical shift) Spinning rate = on the order of CSA linewidth = khz 19 41.7 MHz Solid state 207 Pb spectra Pb(NO 3 ) 2 (no attached protons) Static powder spectrum MAS spectra, spinning rate 3.2 khz 20
Dipolar coupling Local fluctuations at a nuclei, I, will cause a fluctuation of the local field sensed by nuclei S. B 0 θ The size of the local field at nuclei S has an angular dependency given by: I r IS B loc =±μ I r IS -3 (cos 2 θ-1) S In isotropic this interaction is avergaed to zero, but is strong in solids 21 B loc =±μ I r IS -3 (cos 2 θ-1) (where μ = γi) The dipolar interaction can be removed by magic angle spinning. However if a nuclei is attached to a proton we can not spinn at a rate sufficient to remove this interaction due to the large magnetogyric ratio of protons (we would need at least 100 khz spinning rate), then we can instead utilize proton decoupling. 22
23 Quadropolar interaction If the nuclear spinn > ½, the nuclear charge distribution will not be spherical, and the nuclei will possess an electric quadropolar moment. This will have profound effects on NMR signals: 1)Signals may be split into several components 2)Relaxation is very efficient 24
What can NMR tell us about proteins/enzymes? Protein folding Dynamics Interaction surfaces Structure determination (Wolf-Watz et al, 2001, Biochemistry 40, 11423) 25 Proteins are polymers Proteins are polymers built up from amino acid residues. To observe NH groups we must replave the natural 14 N isotope for 15 N. This is accomplished by expressing the protein with E coli. Bacteria is grown in a defined media enriched in thei 15 N isotope (usually 15 N NH 4 Cl). 26
HSQC fingerprint spectrum of an enzyme Each peak corresponds to one amino acid residues proteinet 15 N (ppm) 1 H (ppm) With NMR we can study properties of individual atoms in proteins!!!! 1 H 15 N A pulse sequence is a combination of pulses and delays. A 1 D NMR experiment The 2D 1 H- 15 N HSQC experiment Irradiate 1 H Detection followed By F.T. Transfer magnetization to 15 N. Label with 15 N Chemical shift Back transfer to 1 H
Protein function is ultimately defined by the three dimensional structure Potassium channel in complex with three potassium ions reveals the basis for selectivity towards sodium Rod MacKinnon 1998 Nobel Prize 2003 NMR can be used to solve the structure of proteins in solution, compare to Xray crystallography where the protein is in a crystalline state. This is a bundle of 25 structures calculated primarily using NOE restraints. One of the structures shown with a ribbon representation. (Wolf-Watz et al, 2001, Biochemistry 40, 11423)
Adenylate kinase (Adk) Adk is a small phosphotransfer enzyme that catalyzes the reversible reaction ATPlid ATP+AMP Mg 2+ 2ADP AMPbd Source; Schulz et al., Structure, (1996),15, 147-156. Source; Müller, Schulz J. Mol. Biol. (1992), 224, 159-177. The 2.6 Å structure of thermoadk thermoadk, Henzler-Wildmann, (2007), Nature, 450, 838-844 mesoadk, Schulz et al., Structure, (1996),15, 147-156
Catalytic turnover as f(t) mesoadk thermoadk Ten-fold difference in activity @ room temperature k cat [s -1 ] Strong degree of homology in the active site makes this difference non-intuitive Is this observed difference related to dynamics?? Temperature [C] Opening of sub-domains is rate limiting for catalysis as shown with relaxation (R 2 ) measurements 1570 s -1 1380 s -1 45 s -1 282 s -1 thermoadk k cat = 30 s -1 mesoadk k cat = 290 s -1 Wolf-Watz et al., 2004, Nat. Struct. Biol. 11, 945-949
Development of drugs/inhibitors There exist different methods to use NMR for rational development of drugs binding to proteins. One example is SAR (structure activity relationship) by NMR. Screening for binding is done by looking at interaction surfaces. Step 5 improves the K d significantly by decreasing the entropy of the system. Why study enzymes with NMR? Biofuels Washing powder enzymes Subtilisin Enzymes and disease Energy balance in cells ALS superoxide dismutase ATP + AMP 2ADP ATP is the, Nobel price1997 Adenylat kinase 36
37 The T 2 relaxation affect the appearance of the NMR signal i.e. change the line width Small impurities usually have sharper lines in a protein spectra! 38
The ensemble creates a net magnetization along the z-axis 39