In the next issues, we will comment on how to set up different experiments to analyse protein-ligand interactions by using NMR spectroscopy. First, we will focus on experiments that monitor the recognition process from the ligand’s viewpoint, starting with the TRNOESY experiment, which allows the conformation of a particular ligand at the receptor’s binding site to be deduced.
Sample Preparation and Experimental Set-up of Exchange Transfer NOESY Experiments
As standard, 0.5 mL of 0.05-1 mM of the receptor (protein) is used in this experiment. The ligand is usually present in 5-50 fold molar excess over the concentration of protein binding sites. The selected ligand/protein ratio depends on the binding affinity and on the kinetics of the exchange reaction. If possible, three or four molar ratios should be tested. Although depending on the chemical nature of the ligand, usually non-exchangeable H-C protons are observed. Therefore, the protein samples are deuterated using a regular buffer, i.e., phosphate or deuterated Tris or acetate in deuterated water. In this case, the concentration and deuteration of the protein exchangeable hydrogen atoms may be achieved through repeated lyophilization after suspension in D2O, provided that the protein remains active under these experimental conditions. Otherwise, ultra filtration or dialysis protocols will have to be employed. The experiments can also be performed in regular water (H2O) but then additional pulse modules to remove the very intense H2O signal (more than 100 M concentration) have to be used, as for instance, the NOESY-WATERGATE experiment. [M. Piotto, V. Saudek and V. Sklenar, J. Biomol. NMR , 2 , 661-665 (1992).]
It is strongly advisable that all ligand, receptor and solvent components should also be cleaned passing through a micro-column containing Chelex to remove traces of paramagnetic ions. Other cation binding tags, such as EDTA can also be employed, providing that the binding ability of the receptor is not calcium-dependent.
First, specific 1HNMR spectra of the protein and ligand are separately recorded and analysed. Secondly, the protein sample is titrated using a concentrated stock solution of the ligand in the corresponding buffer. The reverse procedure, employing instead the stock solution of the protein could also be possible, but is not very practical for a variety of reasons. [J. Feeney, J. G. Batchelor, J. P. Albrand and G. C. K. Roberts, J. Magn. Reson . 33 , 519-525 (1979)] The possible existence of chemical shift perturbations, together with evident changes in the signal line widths, are carefully monitored to assess binding for the different ligand/protein molar ratios. Another relatively quick and helpful manner to monitor the interaction is the use of saturation transfer difference experiments [M. Meyer and B. Meyer, Angew. Chem. Int. Ed. Engl., 38 , 1784-1788 (1999)] (forthcoming article). Dissociation rate constants (koff) can be estimated in a qualitative manner, from the dependence of the line width (at half-maximum height) on the ligand/protein ratio. Otherwise, other indirect methods can be used to measure both koff (e.g. surface plasmon resonance) and/or the association constant (e.g. microcalorimetry). Although it is not strictly necessary, the independent assessment of these parameters allows a more quantitative analysis of the obtained TR-NOE data to be performed.
TR-NOESY experiments should be performed using different mixing times (from 25 to 200-400 ms) and ligand/protein ratios. Therefore, several days are typically required to obtain the data. For every ligand/protein ratio, a minimum number of fids (at least 128-256, as in the regular 2D-NOESY) have to be recorded to achieve the proper digital resolution in the evolution dimension (f1), which can be further expanded using linear prediction algorithms. The number of scans is dictated by the amount of material available and by the sensitivity of the NMR instrument. The relaxation delay should be chosen according to the ligand T1 values, and are usually between 2 and 5 seconds. Depending on the size of the protein, mixing times between 25 and 400 ms are used to obtain the TRNOE build-up curves. The data acquired with the longer mixing times will display spin diffusion effects. However, since the initial slope of these curves is less prone to be affected by spin diffusion, it is, therefore, employed for the analysis of the data. Auto- and cross-peak intensities are calculated from the volume integrals or from individual 1D slices of the 2D dataset using commercial software packages. Then, the specific proton pair cross-relaxation rates are estimated from the initial slopes of the time-dependent NOEs, from the ratios of the cross to autopeak intensities at a given mixing time or by fitting the data obtained at the different mixing times to a double exponential equation. [D. Neuhaus and M. P. Williamson, The Nuclear Overhauser Effect in Structural and Conformational Analysis , 2 ed. John Wiley & Sons, New York, (2000).]
It is essential that the binding ability or the activity of the protein is monitored before and after performing the TRNOE experiments. For extracting robust conclusions, the data collected after a significant loss of binding or specificity should be used with extreme caution, since the architecture of the binding site and thus the conformation of the bound ligand could have changed.
The NMR instrument to be employed depends on a variety of factors: First, and more important, on the accessibility to a high-field spectrometer, always depending on the complexity and the degree of overlapping of the 1H NMR spectrum of the ligand. The potential overlap between key protons should be scrutinized. The solubility of the ligand and receptor in the corresponding buffer also plays a key role, as well as the available amount of material for the experiments. For small ligands below 1000 MW, a spectrometer running at 400-500 MHz could be sufficient. The sensitivity of the experiment (and the cross-relaxation rates between protons) increases at higher magnetic fields. Therefore, the availability of very high-field magnets and especially the possible access to instruments equipped with cryo-probes allows TRNOE experiments to be performed with minute amounts of sample. For instance, nowadays, the use of cryo-probes allows protein amounts to be used in the nmol range.
It is necessary to take into account that the assumption of positive cross peaks for the free ligand and negative for the bound one is an oversimplification [V. Roldos, F. J. Cañada and J. Jiménez-Barbero, ChemBioChem , 12, 990-1005 (2011)]. For ligands of MW above 2000, the NOESY cross peaks can already be negative at 500 MHz and room temperature. For larger ligand molecules or at lower temperatures, or at higher magnetic fields, the free ligand will already show negative cross peaks. Thus, the contribution of the free ligand to the observed TRNOE cross peaks should be considered, and the data analysis using a full relaxation matrix approach with chemical exchange is highly advisable.