Interaction studies of small molecules with BC2L-C by Saturation Transfer Difference (STD) NMR

Structural information pertaining to the interactions between biological macromolecules and ligands is of potential significance for the understanding of molecular mechanisms in biological processes. Recently, nuclear magnetic resonance (NMR) spectroscopic techniques have come of age and have widened their scope to characterize binding interactions of small molecules with biological macromolecules especially, proteins. NMR spectroscopy-based methods are versatile due to their ability to examine weak binding interactions and for rapid screening of the binding affinities of ligands with proteins at atomic resolution.
STD-NMR is a simple and fast method for Ligand Screening and characterization of protein-ligand binding near-physiological conditions. It is now frequently used to assess the affinity and specificity of interactions, to identify binding epitopes on proteins and ligands, and to characterize the structural rearrangements induced by binding.
Saturation transfer difference (STD) NMR has developed as one of the foremost prevalent ligand- based NMR methods for the consideration of protein−ligand interactions. The success of this procedure may be a result of its strength and the reality that it is centered on the signals of the ligand, without any requirement of handling NMR data around the receptor and as it was utilizing small quantities of the non-labeled macromolecule.
If a specific proton resonance belonging to a molecule is selectively saturated, this saturation can be transferred to other sites close in space (within ca. 5 Å) to the original proton resonance being saturated. This phenomenon is called "saturation transfer" and is related to the Nuclear Overhauser Effect (NOE).
The experimental sequence requires the acquisition of two different spectra, from which a final difference spectrum is obtained. The first NMR spectrum recorded is the so-called on-resonance (STDon) spectrum, in which the target is selectively saturated by irradiating at a frequency at which the ligand does not resonate (but only the protein protons can be excited). Usually, the irradiation frequencies are chosen around 0 ppm (between 1 and -1 ppm, since it is known from the literature that 92% of the protein binding sites contains at least one aliphatic residue such as Ala, Ile, Leu, or Val, that can be irradiated with a such radiofrequency) or around 8 ppm (to irradiate the aromatic residues of the protein if the ligand does not have aromatic groups). A saturation delay time of the order of seconds allows the target to transfer the excitation by spin diffusion throughout its proton network (i.e., all over the protein) and also to the protons of the ligand next to the protein in the bound site. The rapid exchange between the ligand in the bound state and in the free state leads the saturated ligand to move into the solution where it can be detected.
The second NMR spectrum is the off-resonance (STDoff ) reference spectrum, where the selective excitation is set at a frequency far from the protein signals (around 30 ppm). The off-resonance spectra may be a sort of control and should be the same as a standard proton spectrum of the ligand; in fact, in case the ligand is in excess over the target, only the ligand resonances are visible within the spectrum because it is for the on-resonance spectrum. The saturation of the bound- ligand protons within the on-resonance range leads to a change of their signal intensity.
Difference (STDdiff ) spectrum is obtained by subtracting the on-resonance and off-resonance spectra, in which ligand signals appear with different intensities: the greater the intensity, the greater the proximity of the proton to the protein (Figure 9).
Other compounds that may be shown but without binding activity do not appear within the difference spectra since they do not get any saturation exchange from the receptor. [...]