1) NMR is a method of chemical analysis. (Who uses NMR in this way?) 2) NMR is used as a method for medical imaging. (called MRI )

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1 Uses of NMR: 1) NMR is a method of chemical analysis. (Who uses NMR in this way?) 2) NMR is used as a method for medical imaging. (called MRI ) 3) NMR is used as a method for determining of protein, DNA, RNA structure. (one of only 2 possible methods for doing this)

2 Plan for today - 1) Some basic principles of NMR. 2) NMR as a protein (and DNA and RNA) structure determination method.

3 Nuclei have mass, nuclei have charge. Some nuclei have a property called spin or intrinsic angular momentum. Define: nuclear spin quantum number I magnetic quantum number m. Nuclei with spin have I > zero.

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7 frequency Each H in the protein is in a unique magnetic environment, and can absorb (or emit) radio waves of a unique frequency.

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9 Some simple biochemical applications of NMR.

10 Is the protein folded?

11 NMR can be used for enzyme assays + Spermidine-spermidine acetyltransferase (SSAT) + 1 H NMR spectra can show reactants being converted to products confirms activity of the SSAT enzyme (see next page).

12 spermine SSAT N 1 -acetyl spermidine SSAT N 1,N 12 -diacetyl spermine AcCoA CoA AcCoA CoA

13 Next go to structure determination by NMR..

14 Structural information is derived from NMR data (primarily) through measurement of the Nuclear Overhauser Effect or NOE. NOE = the change in the intensity of the NMR signal of one nucleus when the sample is irradiated with radiowaves at the NMR absorption frequency of another nearby nucleus. The NOE depends on the distance between nuclei.

15 One-dimensional NMR spectra are not useful for measuring the NOE in proteins, due to overlap in the 1 H spectra. It was necessary to invent two-dimensional (2-D) NMR to measure the NOE (and hence 1 H- 1 H distances) in proteins.

16 Illustrate the 2-D NOE spectrum using asparagine as an example.

17 2-D NOE spectrum of a protein with 143 amino acids:

18 In summary: The 2-D NOE intensities provide information from which the distance between protons can be derived. How are NOE intensities converted to distances? Most commonly, a range of distances is used, something like: How are these distances calibrated? Usually by looking at NOE intensities in regular secondary structure, such as helix, where the distances are well known.

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22 What goes into determining a protein structure from NMR data? A medium sized protein may contain 150 amino acids, and > 2000 atoms. NMR data may provide the distances between 1000 pairs of 1 H nuclei (from measured NOE peaks). Are H- 1 H distances enough to determine the structure of this protein? Nope. What to do?

23 What else do we know about the protein structure, in addition to the H- 1 H distances? Bond lengths can be assumed known. Many bond angles are known (tetrahedral carbons, planar rings). Van der Waals radii of atoms are known. How can these different types of information be combined to yield a structure?

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25 Input for simulated annealing: 1) Initial model of the structure (does not need to be correct), with x,y,z coordinates for each atom. 2) Tables containing target values for: a) covalent bond lengths b) bond angles c) van der Waals restraints d) NMR-derived distance restraints (from NOE spectra) e) H-bonds may be included Output from simulated annealing: A new model for the structure (with x,y,z for each atom) which is a better fit to the information 2a) - 2e) above.

26 A starting model for the simulated annealing: Energy function E is very large, due to large violations of NOE-derived distance restraints.

27 Typical result of the simulated annealing process used for protein structure determination: This is a set of 12 super-imposed structures, all of which fit the NMR- derived distance restraints equally well. In other words, these 12 structures all have the same value of E after the simulated annealing process.

28 Visualizing the protein structure.

29 (over about 40 kda spectra are too complex to interpret)

30 With x-ray crystallography, the structure can be directly visualized. Compare this with NMR, where a family of possible structures is derived, each consistent with the observed NMR data.

31 NMR can provide information about which parts of a protein are most rigid, and which are most flexible. The rates at which amide (N-H) protons exchange with the solvent H 2 O provides information about H-bond opening frequencies. H 2 O H H

32 N-H protons that are protected from exchange with solvent can be identified in NMR spectra obtained after transfer of the protein to D 2 O solvent. 1 H - 15 N 2-D spectrum obtained 14 minutes after transfer of protein to D2O solvent.

33 Times for backbone N-H protons to exchange with solvent protons, measured by NMR: Red: milliseconds Yellow: seconds Green: minutes Blue: hours to days Hydrogen bonds will protect N-H groups from exchange with solvent, so slow exchange indicates the presence of hydrogen bonds that open infrequently (located in relatively rigid parts of the protein).

34 An example of protein structure determination by NMR.

35 Illustrate the NMR structure determination using an example: NMR analysis of a protein called Antizyme, a protein inhibitor of ornithine decarboxylase (ODC). Antizyme binds and inhibits ODC, and targets ODC for degradation.

36 How I became involved with antizyme. Dr. Marv Hackert Univ. of Texas

37 Crystal structure of ornithine decarboxylase bind & inhibit Almrud, Oliveira, Kern, Grishin, Phillips & Hackert. JMB, 295, 7-16 (2000).

38 The structure of antizyme: Crystallography or NMR? 1 H NMR spectrum of antizyme from rat, residues ppm

39 Before the structure can be determined, it is necessary to assign the peaks in the 2-D NOE spectrum to specific pairs of 1 H. This is the assignment problem.

40 3-dimensional triple-resonance Fourier Transform-NMR was used for solving the spectrum assignment problem. What is a 3-D NMR spectrum?

41 3-D HNCA spectrum. C 3-D spectrum, with H on first axis, 13 Cα on 2nd axis, and 15 N on 3rd axis. H N15 = ppm plane

42 N15 = ppm plane C H

43 Eventually the assignment of the whole protein backbone is obtained. Other triple-resonance spectra are used to assign additional backbone and side chain nuclei:

44 Eventually generate a table of the NMR frequencies of (almost) all the nuclei in the protein: etc.

45 Once spectrum assignments are complete: Structure determination is performed using Nuclear Overhauser Effect (NOE) spectra, to find protons that are near each other in the structure.

46 2-D NOE spectrum. Too much overlap! Use 3-D NOE spectrum instead, to reduce overlap.

47 3-D NOE spectrum. N ppm ppm ppm ppm ppm N15 = ppm plane

48 NOE peaks between these nuclei in an antiparallel beta strand:

49 NOE peaks between these nuclei in antiparallel beta strands:

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51 Preliminary model of structure: QuickTime and a TIFF (LZW) decompressor are needed to see this picture.

52 Use simulated annealing to produce a refined structure from NMR data:

53 Result of the simulated annealing process used for protein structure determination: This is a set of 12 super-imposed structures, all of which fit the NMRderived distance restraints equally well. In other words, these 12 structures all have the same value of E after the simulated annealing process.

54 Once the structure of Antizyme is known, how do you identify candidates for which parts of the protein are functionally important? (In this case, functionally important means the amino acids that are involved in binding and inhibiting the enzyme Ornithine Decarboxylase). Look at sequence alignment figure to find conserved surface a.a.

55 Blue = conserved, inside protein ; Red = conserved, on surface of protein.

56 Structure, plus locations of conserved amino acids, leads to hypotheses regarding which a.a. may be directly involved in binding to ornithine decarboxylase.