1. A. S. Edison University of Florida 2006 Today’s Lecture 13) Mon, Oct 30: Assignments: I a. Important homonuclear (e.g. 1 H) experiments b. Small molecules c. Peptide assignments

2. A. S. Edison University of Florida 2006 Homonuclear 2D Experiments NameType of correlationNotes COSYCorrelated Spectroscopy 1 H atoms separated by 2 or 3 covalent bonds and mediated by J-coupling (sometimes up to 5 bonds). Several variants available. Only pairs of coupled atoms (compare to TOCSY). Cross peaks antiphase. TOCSYTotal Correlation Spectroscopy 1 H atoms separated by 2 or 3 covalent bonds and mediated by J-coupling. All 1 H in a network of coupled spins will be correlated Correlations between networks of coupled spins. Very useful for side-chains in peptides/proteins. Cross peaks in phase. NOESYNuclear Overhauser Effect Spectroscopy 1 H atoms up to ~5 Å in space mediated by dipolar interactions. The NOE is highly dependent on molecular correlation time: Small molecules have positive NOE and large molecules are negative. Larger molecules have faster transfer rates. Intermediate sized molecules (~500 Da) do not work with NOESY. Crude but important measure of atomic distances; generally categorize into “strong” ( 4 Å). Spin diffusion can cause problems in accurate measurements. ROESYRotating Frame Overhauser Effect Spectroscopy 1 H atoms up to ~5 Å in space mediated by dipolar interactions. Unlike NOESY, ROESY is the same sign regardless of correlation time. This is especially useful for intermediate MW where NOESY doesn’t work. ROESY uses a transverse spin lock and is very similar to TOCSY. For this reason, ROESY experiments often have more artifacts than NOESY.

3. A. S. Edison University of Florida 2006 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. A. S. Edison University of Florida 2006 TOCSY: 60 ms DIPSI-2 mixing time

5. A. S. Edison University of Florida 2006 ROESY: 400 ms cw mixing time

6. A. S. Edison University of Florida 2006 TOCSY and COSY comparison

7. A. S. Edison University of Florida 2006 ROESY and COSY comparison

8. A. S. Edison University of Florida 2006 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 I x S x and I y S y terms. The net result of the TOCSY mixing sequence is the transfer of magnetization from I to S along the same axis (e.g. I z to S z or I x to S x or I y to S y ). 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. A. S. Edison University of Florida 2006 TOCSY Pulse sequence (the simplest of several variants) A. S. Edison University of Florida 90x t1t1 Isotropic Mixing 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 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)

10. A. S. Edison University of Florida 2006 Spin Systems CH 3 HHHHHH HHHH H 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. A. S. Edison University of Florida 2006 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. A. S. Edison University of Florida Zachariah, C., Cameron, A., Lindberg, I., Kao, K. J., Beinfeld, M. C., and Edison, A. S. “Structural Studies of a Neuropeptide Precursor Protein with an RGD Proteolytic Site” Biochemistry 40, 8790-8799 (2001).

12. A. S. Edison University of Florida 2006 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 T 1 (longitudinal or spin-latice) or T 2 (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. A. S. Edison University of Florida 2006 A. S. Edison University of Florida NOE vs. ROE

14. A. S. Edison University of Florida 2006 A. S. Edison University of Florida NOESY pulse sequence A. S. Edison University of Florida 90    t1t1 90   90   Like all 2D sequences, t 1 is the variable time to collect frequency information in the indirect dimension. The delay  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  ’s make up a phase- cycle, and we can also adjust which axis from which to record the signal (  rec ). These are designed to minimize artifacts and select for desired correlations. Most 2D pulse sequences have phase cycles.  rec

15. A. S. Edison University of Florida 2006 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. Notice the difference between TOCSY (black) and NOESY (red).

16. A. S. Edison University of Florida 2006 TOCSY/COSY NOESY/ROESY 1 H-based Peptide Resonance Assignments N C  C’ N C  H O H O H CH 3 H 3 3 N H

17. A. S. Edison University of Florida 2006 1 H-based Resonance Assignments F2 G3 E5 G10 M6 M8 F14 V11 D4 L12 R13 "NH" 2 8.78.48.17.8 ppm 4.8 4.5 4.2 3.9 GFGDEMSMPGVLRFamide S7

18. A. S. Edison University of Florida 2006 Next Lecture A. S. Edison University of Florida 14) Wed, Nov 1: Assignments: II a.Important heteronuclear experiments b.3D NMR c.Assignment strategies in proteins d.Intro to protein structure determination