Fast Course in NMR Lecture 7 Jan/Feb, 2016 Heteronuclear 2D NMR Elements of heteronuclear sequences HSQC HMQC

Building Blocks There are large numbers of 2D and 3D NMR heteronuclear experiments. However, most of the experiments can be broken down to a few very simple “building blocks”. The most fundamental building block is the spin echo sequence. 90x D 180x Case 1: No coupling. Chemical shift is refocused during the period 2D.

90x 180x Building Blocks D I: Case 2: Coupling. We just showed that chemical shift is refocused during the period 2D. It can easily be shown that this holds with an I-S coupled pair of spins. Therefore, we will ignore the chemical shift operator during the symmetric D periods. This refocusing period also decouples I from S.

Building Blocks D 90x 180x I: S: Case 3: Coupling. With both 180’s, the spins stay coupled for the entire period 2D.

Building Blocks D 90x 180x I: S: INEPT: Insensitive nuclei enhancement by polarization transfer. Adjust D to 1/(4*JIS):

Building Blocks D 90x 180x 90y I: S: INEPT: Insensitive nuclei enhancement by polarization transfer. Adjust D to 1/(4*JIS): INEPT transfers the magnetization from I to S. Notice that following the 2 simultaneous 90 degree pulses, the S term is in the x-y plane.

(Heteronuclear Single Quantum Correlation) HSQC (Heteronuclear Single Quantum Correlation) 90x D 180x I: S: 90y t1/2 Decouple Adjust D to 1/(4*JIS):

(Heteronuclear Multiple Quantum Correlation) HMQC (Heteronuclear Multiple Quantum Correlation) 90x D I: S: t1/2 180x 90(x,-x) Decouple Adjust D to 1/(2*JIS): x,-x Self Study: Evaluate this sequence using product operators

HSQC: phase cycles 90x D 180x 90y 90f1 180f2 90f3 frec I: S: Decouple t1/2 180f2 90f3 Decouple Adjust D to 1/(4*JIS): frec f1=x, -x f2=4y, 4(-y) f3=2x, 2(-x) frec=2(x, -x, x, -x) The phases of some pulses are varied in a phase cycle. This is done for different reasons. In the above example, the phase for f1 and f3 changes the sign of the desired magnetization. Therefore, the receiver phase, frec, is adjusted so that the desired magnetization is added together. The phase, f2, does not change the sign of the desired magnetization, so the receiver doesn’t change with it. f2 is there to eliminate artifacts due to a pulse that is not exactly 180 degrees.

Why Heteronuclear NMR? Beyond relatively small peptides, most NMR of proteins or nucleic acids requires isotopically labeled samples. Proteins are usually produced by over-expressing them in bacteria with 15N-labeled, 13C-labeled, 15N+13C-labeled, or 15N+13C+2H-labeled nutrients. Specific labeling strategies have been developed, but uniform isotopic enrichment is most common. There are several reasons to isotopically label a sample: Overlapping 1H resonances can be resolved using the frequencies of the attached heteronucleus. The heteronuclei create efficient pathways to correlate atoms along the backbone and side-chains of proteins and nucleic acids. The heteronuclei provide sites to probe dynamics of the molecule by examining their relaxation properties. Selective labeling of different molecules allows mixtures to be easily studied (e.g. ligand binding). Transverse Relaxation Optimized Spectroscopy (TROSY) utilizes heteronuclei and allows proteins as large as 1 million Daltons to be studied.

15N-HSQC correlates 1H and 15N Each amide group in a protein gives rise to a peak in a 2D spectrum.

15N-HSQC of IA3, an unfolded protein. Green, T. Ganesh, O., Perry, K., Smith, L., Phylip, L. H., Logan, T. M., Hagen, S. J., Dunn, B. M., Edison, A. S., “IA3, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, Is Intrinsically Unstructured in Solution” Biochemistry 43, 4071-4081 (2004).

HSQC can determine whether a protein is folded or not 6.0 7.0 8.0 9.0 10.0 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 1H (ppm) 15N (ppm) Coil 1: Ubiquitin Coil 2: IA3

13C-HSQC of anisomorphal: a monoterpene 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).

HSQC: Watergate 180 90x 180x 90y 180f2 90x 180x frec D D D D d d 180x I: 180x 90f1 90f3 180x D D D D t1/2 t1/2 S: Decouple Gradient: Gradients allow the magnetic field to be changed linearly along a given axis (x, y, or z). Many modern NMR experiments utilize gradients to either wipe out unwanted magnetization or, as an alternative to phase cycling, to keep wanted magnetization. The Watergate sequence is a very nice alternative to presaturation to remove water from an NMR spectrum. It can be placed at the end of many different sequences and is very simple, consisting of 2 gradient pulses (red) and a selective 180 degree pulse (blue).

Watergate 180 frec d d I: S: Decouple Gradient: The Watergate sequence takes transverse magnetization that is otherwise ready to detect. Then, the magnetization goes through a modified spin echo sequence. The gradients change the magnetic field across the sample, for example causing spins at the top of the tube to have a greater frequency than those in the bottom. The 180 degree pulse is designed to invert everything except water (which is usually on-resonance). Any spin that is inverted will be refocused by the second gradient pulse. Any spin (e.g. water) that is not inverted by the 180 degree pulse will be further defocused with the second gradient. A good and popular way to achieve this is with a “3-9-19” pulse:

Triple Resonance: HNCA 90x D 180x 1H 180f1 15N 90f2 t1/2 180f4 Decouple frec 13Ca 90f3 D` 13CO t2/2 Most “triple-resonance” experiments transfer magnetization through single bond couplings. The working parts of the experiments are often just INEPT-like transfers from one spin to another. For example, in the HNCA experiment, the first step is an HSQC from 1H to 15N. Rather than go directly back to 1H, a second HSQC transfers the magnetization to the 13Ca, which is allowed to evolve in a second indirect time period, t2. The net result is a signal as a function of 3 frequencies, 1H(t3), 13Ca(t2), and 15N(t1). See Cavanagh et al. for more detail.