Triple-resonance experiments

Many different types of 3D and 4D experiments can be devised. We described briefly one class, in which two different kinds of 2D experiments are joined, primarily to use the third dimension to clarify the presentation of spectral data. With the ability of most modern NMR spectrometers to provide precise frequency and phase control for several radio frequencies simultaneously and to apply them efficiently in the probe, a class of 3D and 4D triple resonance experiments has become feasible. These experiments usually use two HSQC and/or HMQC sequences to transfer magnetization in the path I— S— T— S— I. I is almost always H 5 and T are 13C and 15N in proteins, the type of molecule in which these experiments are most often utilized. In addition,31P may be involved in nucleic acids, another frequent subject for these methods, and many other nuclides can be used in other applications. 

As an example, we describe one of many similar experiments devised for assigning resonances in proteins, a subject that we take up in more detail in Chapter 13. This particular experiment is designed to correlate the frequencies within the H—15N—13C=0 portion of a peptide group and is appropriately called simply HNCO. The basic pulse sequence for HNCO is shown in Fig. 12.16. To simplify the notation, instead of I, S, and T, we identify the active spins as H, N, and C, and use K to denote the spin of ar-13C. In a peptide chain, one a carbon is bonded and spin coupled to the nitrogen and another a carbon is bonded and coupled to the carbonyl carbon atom. With recombinant DNA methods, the protein is uniformly and highly enriched in both 13C and 15N, so all of these spins need be considered. 

Next comes a fixed period A = 1/2 JNC, during which N is influenced by its chemical shift and all couplings except that from K, which is removed by the 

FIGURE 12.16 Pulse sequence for the triple resonance 3D NMR experiment HNCO. H and N denote H and 15N, C denotes 13C=0, and K denotes 13C . Pulses at times 1, 2, and 3 constitute an INEPT sequence that transfers coherence from H to. V, where it precesses during q. Pulses at times 6, 7, and 8 represent an HMQC sequence that creates multiple quantum coherence in C (where it precesses during and transfers coherence back to N. Pulses 10 and 11 are an inverse INEPT sequence that transfers coherence back to H for detection during f3.The other 180° pulses refocus heteronuclear spin couplings. Note that coherence is not transferred to spin K. 

180° K pulse in the middle of the A period. However, the N 180° pulse in the middle to the later t2 period will eventually refocus the N chemical shifts and JNH, so we can concentrate on the effect of JNC during the A period. As we have seen previously, evolution of N magnetization for a period 1/2JNC develops N magnetization that is antiphase in C.Thus, the initial term HzNy becomes HZNXCZ, and the first C 90° pulse converts this to DQC and ZQC, as we have seen for HMQC  

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All P.M.R. spectra were measured with a Varian HA 100 spectrometer operating in the frequency-sweep mode with tetramethylsilane as the reference for the internal lock. The double and triple resonance experiments were performed using a Hewlett Packard 200 CD audio-oscillator and a modified Hewlett Packard 200 AB audio-oscillator (vide infra). Spectra were measured using whichever sweep width was required to ensure adequate resolution of the multiplets under investigation, generally 250 or 100 Hz, and sweep rates were selected as necessary. Extensive use was made of the Difference 1 and Difference 2 calibration modes of the instrument, both for the decoupling experiments and for the calibration of normal spectra. 

Figure 3 3-D and 4-D triple resonance experiments correlate interresidue or... 

Lohr F, Riiterjans H. A triple resonance experiment for the sequential assignment of backbone resonances in proteins. J Biomol NMR 1995 6 189-197... 

While the CA and CO secondary chemical shifts cannot be determined using conventional triple-resonance experiments that employ the H-15N correlation spectrum, incorporation of individual... 

Let us first discuss common coherence transfer steps of triple-resonance experiments with the emphasis on relaxation mechanisms. 

Exploitation of the TROSY effect is rather straightforward. In contrast to 15N-HSQC (Heteronuclear Single Quantum Coherence) or standard triple-resonance experiments based on 15N-HSQC, no radio frequency pulses or composite pulse decoupling should be applied on amide protons when HN spin is not in the transverse plane. Likewise the 15N decoupling should be... 

At this point, before going into the details of triple-resonance experiments, we shift the focus to the polarization transfer step. Throughout this... 

Figure 3 depicts the famous triple-resonance experiment, HNCA (amide proton to nitrogen to alpha carbon experiment)10,32 34 (Fig. 3a), and the... 

The effect of deuteration, for the needs of sequence-specific resonance assignments using triple-resonance experiments, was first demonstrated by Bax and co-workers on the 19.7 kDa protein calcineurin B in 1993.56 By replacing the H spin with deuterium, the transverse relaxation time of 13C spin is increased by nearly an order of magnitude due to the 6.5 times smaller gyromagnetic ratio of 2H in comparison to in.52,56 Not surprisingly, deuteration was utilized for the aid of structure determination of several... 

Obtaining good quality NMR spectra of large molecular structures is an essential first step. However, in addition, the assignment of the chemical shifts to individual nuclei is indispensable as a basis for detailed structural studies. Sequential assignment is achieved with triple resonance experiments [28] (chapter 4). In these experiments, magnetization is transferred between 1HN, 15N and 13C, and they are routinely applied with molecular sizes up to about 30 kDa. The use of the [ N HJ-TROSY in triple resonance experi-... 

Fig. 10.5 Sequential resonance assignment of the polypeptide backbone of 2H, 3C, 5N-labeled DHNA using the HNCA triple resonance experiment, which connects the Hn and 15N resonances of the amide groups with the sequential and intraresidual 13C chemical shifts. The dotted...

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