Saturation Transfer Difference (STD) NMR Spectroscopy

Saturation Transfer Difference (STD) NMR is a recently explored form of NMR anal- ysis in which a difference in signal is observed and subtracted between the bound and non-bound ligand and its binding partner.144 If there is an interaction, a signal is ob- served on the difference spectra, as the two signals do not completely cancel out. This technique was first published in 1999 by Mayer et al.145 Their initial experiments in- volved mixtures of ligands for rapid screening and ligand mapping of potential binding compounds. The initial paper focussed on the binding of oligosaccharides and they were even able to characterise GlcNAc as a binder through the proton and STD spectra for this compound. The benefits of this technique include the decreased concentration of target required (compared to techniques such as 15N-HSQC), the relative ease of use of the technique as well as the ability to examine large molecular weight targets.

In 2005, Krishnan et al. produced a comprehensive review stating the details of how NMR can be used to determine protein-ligand interactions. Using the notion that re- ceptors and ligands display NMR-type characteristics at equilibrium, it is possible to analyse their binding modality at this point.144 It is the free resonating receptor and ligand that is NMR-active, whilst the complex itself is NMR-silent. In order to produce a difference in resonance state, a magnetic frequency is applied to the receptor-ligand mixture, which is equal to the resonance frequency of the receptor. This causes the receptor to resonate with greater intensity and resonance travels across the receptor through spin diffusion. It is key to note that there must be no resonance from the ligand, else this would interfere with the resultant difference spectra.

Figure 2.15: Diagrammatic representation of spin diffusion and resonance throughout the target, ligand and a non-binder (star). A. No resonance. B. Application of magnetic field and spin diffusion across the target. C. Spin transfer from the target to the ligand

Once the resonance has spread throughout the receptor it is then transferred to the ligand through dipole-dipole interaction of neighbouring protons. This resonance causes the ligand to dissociate from the receptor and causes fresh free ligand to associate with the target, after which the process is repeated.

A reference “off”-resonance signal is then deduced, which is recorded at a frequency in which neither the receptor nor the ligand resonates (this is usually between 25 and 50 ppm from the on-resonance frequency).

In order to maintain consistency, the temperature of the environment is controlled (in the case of our experiments, each run was carried out at 5◦C, as this not only preserved the life of the protein but also reduced the speed of which the macromolecule tumbled, hence improving the signal intensities and reducing background noise.

Once the “on” and “off” signals are subtracted, the difference signals left are the result of the saturated receptor and ligand and, as both the receptor and ligand are present in solution at their minimum working concentration, the receptor signal is rarely seen. This technique can also be used to determine KD values as multiple experiments can

be setup in parallel with varying concentrations of ligand, whilst the protein is kept constant.

In order to maximise the signal produced, experiments were carried out on an 800 MHz NMR machine (for the greatest possible resolution of the peaks in A34) and in a 300 µL NMR tube to reduce the volume necessary for the experiment. Due to high surface tension, it was vital that components were added to the tube slowly and the tube was later centrifuged in order to force components to the bottom of the tube and the air to the top.

Factors that affect the STD-NMR spectra include the biochemistry of the receptor-ligand interaction, as well as the type of molecules, for example, protons in carbohydrates and in DNA are less capable of spin diffusion as protons are further apart and therefore the transfer of spin is less efficient, hence why proteins are more commonly used, as they rapidly and readily transfer spin between neighbouring protons to saturate the entire macromolecule.146 Larger proteins also tend to work better, as they tend to tumble more slowly in solution and therefore create a greater difference spectrum.147 The working concentrations of protein required are between 10 µM and 20 µM when in the sample, whilst the quantity of ligand is approximately 500 µg. For the purpose of this study, the protein concentration was estimated to be approximately 10 µM and the quantity of ligand to be 500 µg.

Additional factors to consider when using this technique includes the KD of the ligand,

pulses will not detect a difference between the free and bound states, as any changes in signal would not be detected at the pulse rate of the machine. For this reason, A34 was chosen, as it was a moderate strength binder (Ki = 7.125 µM) and would not bind too

strongly or weakly to the protein.

Another factor to consider includes the buffer system, as the presence of DMSO in the buffer can interfere with the NMR (although interestingly, the presence of DMSO in this case appeared to improve magnetic distribution across the protein). It is also important to note that all components require freeze drying multiple times with deuterium oxide to remove any water present on the molecules that could swamp the NMR signal. Also, it is important to determine the optimum temperature for the experiments, as lower temperatures decrease the tumbling in solution but may improve the stability of the protein for analysis. The buffer used in the analysis was 10% DMSO-d6 (to solubilise

the ligand) in PBS (10 mM PO43, 137 mM NaCl, and 2.7 mM KCl) at pH 7.4 to stabilise

the protein.