3.1.3. Saturation Transfer Difference (STD)
Saturation Transfer Difference (STD) NMR depends on the Nuclear Overhauser Effect (NOE), which is often used to enhance the sensitivity of less sensitive nuclei such as 13C and 15N [256,257]. This increase in sensitivity is possible because of dipolar coupling (i.e., through space interactions of separate nuclei) [257]. The increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [256] of a specific chemical group (i.e., the methyl groups on a protein), which is then given time to transfer to another chemical group via the NOE dipolar coupling within a few angstroms [258]. The transfer in magnetization is easily visualized on a NMR spectrum that takes the differences in the signal intensities from before and after the irradiation. This new spectrum is called a “difference spectrum”, and it reveals what chemical groups interact with the irradiated signal [259] (see Figure 8).
STD NMR is an application of NOE used to probe the binding of ligands to a specific site within the targeted proteins [256]. A generic example of detecting ligand binding via STD is presented in Figure 9a. The STD NMR method follows the same concepts as a normal NOE experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e., methyls) with a saturated signal from a previous selective radiofrequency pulse. The saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. The difference in peak intensities proves the presence of ligand binding [260].
Water-Ligand Observed through Gradient Spectroscopy (WaterLOGSY) is a second type of STD (see Figure 9b). The main difference with normal STD NMR is that water is the saturated signal [261], and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [209].
For STD NMR to work properly, the ligand concentration must be in large excess (often 100–1000 fold) over the receptor so that effective saturation transfer can take place [260]. This means that for STD NMR, and WaterLOGSY, only small amounts (µg) of protein are required to get results [261,262,263]. This is advantageous for researchers, as they can perform STD NMR on a protein of interest, and preserve the rest of the unused sample for future/other experiments. Also, the same sample can be used for multiple NMR measurements. STD NMR facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in Figure 9. WaterLOGSY has been extended to study ligand interactions with DNA and RNA [261].
There are additional NOE-type experiments (trNOE, INPHARMA, SALMON, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [264].
With the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. Benie et al. [265] described the use of saturation transfer difference (STD) NMR spectroscopy [262,266,267,268,269,270,271] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype 2 (HRV2). The experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an HRV2 particle cut open, cf. the table of contents) to STD NMR experiments. The principles of STD NMR have been known for many years [267,268] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [262,266]. The technique also permitted the analysis of epitopes of ligands bound to receptor proteins. Previous NMR studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. This approach was unworkable when studying pathogenic viruses. Benie et al. [265] demonstrated that solution state STD methodology not only reduces the amount of virus required by approximately 2 orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [272].
The very large size of viruses makes them particularly attractive for studies by STD NMR, as they inherently yield large line widths allowing for easy irradiation of the virus without affecting the ligand protons. Furthermore, because of the larger correlation time of a virus in comparison to an average-sized protein, spin diffusion, and thus saturation transfer, is very efficient. The large line width has additional benefits not just for STD-based NMR methods but also for transfer NOESY spectra, as protons from the virus capsid are invisible in the NMR spectra (for an example of a transfer NOESY spectrum, see [265]). Moreover, competitive STD titration experiments can be used to determine the Kd value of a ligand [271]. Analysis of the STD spectra using the group epitope mapping method [271] allows for the determination of the binding epitope. STD NMR methods can considerably speed up the determination of the binding epitope for potential antiviral lead compounds.
Simple STD NMR experiments provide substantial information on the binding of ligands to native viruses and require very small amounts of the virus with measurement times in the range of tens of minutes. This allows for a high throughput of ligand samples without significant consumption of viral material because it remains unaffected by the experiments and is easily separated from the low molecular weight ligands by ultra-filtration subsequently. In addition to the detection of binding, a complete mapping of the ligand-binding epitope can be achieved [265].
Noroviruses (NV) are non-enveloped, single-stranded, positive-sense RNA viruses that are the major cause of epidemic outbreaks of gastroenteritis worldwide [273,274,275]. The viral coat consists of a single protein, VP1, which assembles into a capsid with overall icosahedral symmetry [276,277,278]. Attachment of human noroviruses to histo-blood group antigens (HBGAs) is thought to be critical for the infection process [279]. The protruding domains of the VP1 proteins, called P-domains, harbor highly conserved binding sites for HBGAs. STD NMR-based epitope mapping was used [262,271] to identify structural features of different core types critical for the binding of synthetic A- and B-tetrasaccharides [280] to virus-like particles (VLPs) of a highly homologous GII.4 strain (Ast6139). STD NMR experiments provide a robust and straightforward technique for obtaining ligand binding epitopes at atomic resolution. Comparing binding epitopes of related ligands then delivers critical information about structural requirements for ligand recognition. Conversely, comparison of binding epitopes of a given ligand binding to wild type, and to mutant proteins reveals the importance of individual amino acids for binding. STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes and suggest that these two mutations do not significantly alter HBGA recognition. The STD NMR approach to characterize binding of HBGA ligands to noroviruses has employed VLPs as targets and thus taken advantage of the large size of VLPs yielding excellent signal-to-noise ratios of the corresponding STD NMR spectra, as demonstrated previously [281].