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Today’s Lecture

Today’s Lecture. 13) Mon, Oct 30: Assignments: I a. Important homonuclear (e.g. 1 H) experiments b. Small molecules c. Peptide assignments. Homonuclear 2D Experiments. DQF-COSY: Double quantum filtered COSY to clean up diagonal and reduce solvent.

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Today’s Lecture

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  1. Today’s Lecture 13) Mon, Oct 30: Assignments: I a. Important homonuclear (e.g. 1H) experiments b. Small molecules c. Peptide assignments

  2. Homonuclear 2D Experiments

  3. DQF-COSY: Double quantum filtered COSY to clean up diagonal and reduce solvent Dossey, A. T., Walse, S. S., Rocca, J. R., & Edison, A. S. “Single Insect NMR: A New Tool to Probe Chemical Biodiversity” ACS Chemical Biology, 1 (8), 511–514 (2006).

  4. TOCSY: 60 ms DIPSI-2 mixing time

  5. ROESY: 400 ms cw mixing time

  6. TOCSY and COSY comparison

  7. ROESY and COSY comparison

  8. TOCSY OVERVIEW • TOCSY (Total Correlation Spectroscopy) is capable of correlating all spins in a coupled network (examples shown on the next slide). • The “working end” of the TOCSY pulse sequence is an isotropic mixing sequence that is a sequence of pulses that are designed to remove all chemical shift differences and create a strong coupling environment. This is called the Hartmann-Hahn condition. Sometimes the TOCSY experiment is called HOHAHA (Homonuclear Hartmann-Hahn). • Several mixing sequencings have been developed. The most popular are MLEV-17, WALTZ-16, GARP, and DIPSI. These all apply a series of pulses with either different phases or different lengths or both. • The strong coupling product operator is similar to the regular (weak) coupling operator but it includes IxSx and IySy terms. • The net result of the TOCSY mixing sequence is the transfer of magnetization from I to S along the same axis (e.g. Iz to Sz or Ix to Sx or Iy to Sy). The transfer depends on the coupling constant but is fairly complicated because S can transfer to another spin, R, and so on. A. S. Edison University of Florida

  9. 90x Isotropic Mixing TOCSY Pulse sequence (the simplest of several variants) t1 The numbers are pulse lengths in degrees. The red and black pulses are 180 degrees out of phase. The element in the parenthesis is repeated n-times to get the desired mixing time (e.g. 20-80 ms) MLEV-17: (90-y, 180x, 90-y, 90-y, 180x, 90-y)*n 60x DIPSI-2: (320, 410, 290, 285, 30, 245, 375, 265, 370)*n A. S. Edison University of Florida

  10. H H H H H H H CH3 CH3 H H H H CH3 Spin Systems Any set of protons in a chain of unbroken J-coupling interactions will give rise to sets of TOCSY cross peaks. For example, each of the 3 sets of peaks shown above will be correlated in a 2D TOCSY spectrum. A. S. Edison University of Florida

  11. TOCSY spectrum of a 16 amino acid peptide Each amino acid will produce a pattern of peaks that represent the network of coupled spins in that amino acid. The highlighted spin system shown in red is a leucine. Note that there are two leucines with very similar chemical shifts. Zachariah, C., Cameron, A., Lindberg, I., Kao, K. J., Beinfeld, M. C., andEdison, A. S. “Structural Studies of a Neuropeptide Precursor Protein with an RGD Proteolytic Site”Biochemistry40, 8790-8799 (2001). A. S. Edison University of Florida

  12. NOE • Transferring magnetization through scalar coupling is a “coherent” process. This means that all of the spins are doing the same thing at the same time. • Relaxation is an “incoherent” process, because it is caused by random fluxuations that are not coordinated. • The nuclear Overhauser effect (NOE) is in incoherent process in which two nuclear spins “cross-relax”. Recall that a single spin can relax by T1 (longitudinal or spin-latice) or T2 (transverse or spin-spin) mechanisms. Nuclear spins can also cross-relax through dipole-dipole interactions and other mechanisms. This cross relaxation causes changes in one spin through perturbations of the other spin. • The NOE is dependent on many factors. The major factors are molecular tumbling frequency and internuclear distance. The intensity of the NOE is proportional to r-6 where r is the distance between the 2 spins. A. S. Edison University of Florida

  13. Enhancement NOE goes through zero wtc NOE ~33kDa ~10 kDa Small peptides NOE vs. ROE A. S. Edison University of Florida

  14. 90f2 90f3 90f1 t1 t NOESY pulse sequence frec Like all 2D sequences, t1 is the variable time to collect frequency information in the indirect dimension. The delay t is fixed and is the time during which the NOE builds up. You might have guessed that chemical exchange can also happen during this time, and it is possible to confuse an NOE peak with a chemical exchange peak, but techniques have been developed to figure out which is which. Notice that I have not indicated the phases of the pulses. The different f’s make up a phase-cycle, and we can also adjust which axis from which to record the signal (frec). These are designed to minimize artifacts and select for desired correlations. Most 2D pulse sequences have phase cycles. A. S. Edison University of Florida A. S. Edison University of Florida

  15. Notice the difference between TOCSY (black) and NOESY (red). NOESY and TOCSY spectra of a 16 amino acid peptide Each cross peak in a NOESY spectrum indicates that the nuclei resonating at the 2 frequencies are within 5 Å in space.

  16. H O H CH 3 a C’ C N a C’ C N N H CH H H O CH CH 3 3 TOCSY/COSY NOESY/ROESY 1H-based Peptide Resonance Assignments

  17. GFGDEMSMPGVLRFamide F2 S7 G10 V11 3.9 L12 E5 R13 4.2 4.5 M6 G3 D4 F14 "NH" 2 4.8 M8 8.7 8.4 8.1 7.8 ppm 1H-based Resonance Assignments

  18. Next Lecture 14) Wed, Nov 1: Assignments: II Important heteronuclear experiments 3D NMR Assignment strategies in proteins Intro to protein structure determination A. S. Edison University of Florida

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