Triple resonance experiments

Triple resonance experiments are a set of multi-dimensional nuclear magnetic resonance spectroscopy (NMR) experiments that link three types of atomic nuclei, most typically consisting of ¹H, ¹⁵N and ¹³C. These experiments are often used to assign specific resonance signals to specific atoms in an isotopically-enriched protein. The technique was first described in papers by Ad Bax, Mitsuhiko Ikura and Lewis Kay in 1990, and further experiments were then added to the suite of experiments. Many of these experiments have since become the standard set of experiments used for sequential assignment of NMR resonances in the determination of protein structure by NMR. They are now an integral part of solution NMR study of proteins, and they may also be used in solid-state NMR.

There are two main methods of determining protein structure on the atomic level. The first of these is by X-ray crystallography, starting in 1958 when the crystal structure of myoglobin was determined. The second method is by NMR, which began in the 1980s when Kurt Wüthrich outlined the framework for NMR structure determination of proteins and solved the structure of small globular proteins. The early method of structural determination of protein by NMR relied on proton-based homonuclear NMR spectroscopy in which the size of the protein that may be determined is limited to ~10 KDa. This limitation is due to the need to assign NMR signals from the large number of nuclei in the protein – in larger protein, the greater number of nuclei results in overcrowding of resonances, and the increasing size of the protein also broadens the signals, making resonance assignment difficult. These problems may be alleviated by using heteronuclear NMR spectroscopy which allows the proton spectrum to be edited with respect to the ¹⁵N and ¹³C chemical shifts, and also reduces the overlap of resonances by increasing the number of dimensions of the spectrum. In 1990, Ad Bax and coworkers developed the triple resonance technology and experiments on proteins isotopically labelled with ¹⁵N and ¹³C, with the result that the spectra are dramatically simplified, greatly facilitating the process of resonance assignment, and increasing the size of the protein that may be determined by NMR.

...
Wikipedia