a. s. edison university of florida 2006 today’s lecture 13) mon, oct 30: assignments: i a....

A. S. EdisonUniversity of Florida2006

Today’s Lecture

13) Mon, Oct 30: Assignments: Ia. Important homonuclear (e.g. 1H)

experimentsb. Small moleculesc. Peptide assignments


Homonuclear 2D ExperimentsName Type of correlation Notes

COSY Correlated Spectroscopy

1H 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.

TOCSY Total Correlation Spectroscopy

1H atoms separated by 2 or 3 covalent bonds and mediated by J-coupling. All 1H 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.

NOESY Nuclear Overhauser Effect Spectroscopy

1H 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” (<2.5 Å), “medium” (<4 Å), “weak” (>4 Å). Spin diffusion can cause problems in accurate measurements.

ROESY Rotating Frame Overhauser Effect Spectroscopy

1H 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.


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).


TOCSY: 60 ms DIPSI-2 mixing time


ROESY: 400 ms cw mixing time


TOCSY and COSY comparison


ROESY and COSY comparison


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. EdisonUniversity of Florida


TOCSY Pulse sequence

(the simplest of several variants)


90x

t1 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)


Spin Systems

CH3

CH3CH3

H H H H H H

H H H H

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.



TOCSY spectrum of a 16 amino acid peptide

H2

H1

1357

1

3

5

7

9

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., and Edison, A. S. “Structural Studies of a Neuropeptide Precursor Protein with an RGD Proteolytic Site” Biochemistry 40, 8790-8799 (2001).


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.




NOE vs. ROE

NOE goes through zero c

NOE

Small peptides ~10 kDa ~33kDa

Enh

ance

men

t



NOESY pulse sequence


90

t1

90 90

Like all 2D sequences, t1 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


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.

H2

H1

1357

1

3

5

7

9

Notice the difference between TOCSY (black) and NOESY (red).


TOCSY/COSY

NOESY/ROESY

1H-based Peptide Resonance Assignments

NC

C’N

CC’

H O

H OHCH3

HCH

CH3CH3

N

H


1H-based Resonance Assignments

F2

G3

E5

G10

M6

M8

F14

V11

D4

L12

R13

"NH"2

8.7 8.4 8.1 7.8ppm

4.8

4.5

4.2

3.9

GFGDEMSMPGVLRFamide

S7


Next Lecture


14) Wed, Nov 1: Assignments: II

a.Important heteronuclear experiments

b.3D NMR

c.Assignment strategies in proteins

d.Intro to protein structure determination

a. s. edison university of florida 2006 today’s lecture 13) mon, oct 30: assignments: i a....

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