Binding: Saturation Transfer Difference

One of the strengths of NMR is the variety of methods that have been developed to understand different aspects of molecular interactions. The previous post discussed the waterLOGSY experiment, which identifies small molecules that bind to larger molecules. The saturation transfer difference experiment is another method that identifies molecules that bind another, but it relies on a different mechanism than the waterLOGSY experiment and so is often useful in different situations.

Spectra obtained from a saturation transfer difference (STD) experiment are shown below. The same sample that was used for the waterLOGSY spectra was used here, namely 1.0 mM L-tryptophan, 1.0 mM D-sucrose and 0.1 mM bovine serum albumin (BSA) in phosphate buffer at pH 6.5. The figure shows the two 1D spectra obtained from the standard Bruker implementation of the STD experiment. The upper, red spectrum is a reference spectrum, which shows signals from everything in the sample, while the lower, blue spectrum shows signals only from the molecules that bind.

Comparison of the two spectra shows that the STD spectrum does not contain any of the sucrose peaks. The anomeric sucrose signal near 5.3 ppm is missing, as are the signals between 4.0 and 3.5 ppm. The large sucrose methylene peaks around 3.7 ppm have produced sharp positive and negative subtraction artifacts, but these are relatively easy to distinguish from the real aliphatic tryptophan peaks in the same region.

Where waterLOGSY relies on the NOE to transfer magnetisation between molecules, the STD uses spin diffusion. As the name suggests, STD is a difference experiment. A pair of spectra are collected with saturation at different positions and a difference spectrum generated. In the example above the excitation positions used were -40 ppm, where no resonances appear, and 0 ppm where only resonances of the large molecule (BSA) appear. When the resonances of the large molecule are saturated, then spin diffusion can transfer their magnetisation to nearby nuclei that were not excited. For example, those of a small molecule that is bound to the large one. Generating the difference between the two spectra shows signals only from the nuclei in contact with the large molecule.

The STD and waterLOGSY experiments both do much the same thing, they allow a mixture of molecules to be screened for those that bind to a large target molecule. Their differences, however, complement each other and allow a greater range of systems to be studied. For example STD has no limitations on the size of the molecules being examined, whereas waterLOGSY reports only on small molecules binding large ones. However, waterLOGSY is more sensitive than STD, and can be used even if there is no part of the spectrum that contains only peaks from the large molecule.