As the pressure increases, the intensity of the bound-state peak increases and both peaks propagate along δ( 1H) and δ( 15N) axes in the 1H– 15N HSQC spectra.
The right panels in Figure 4E–H show the behavior of the Tyr 194 peak at different pressures and a fixed concentration of the ligand. Pressure can also be used to rearrange the intensity ratio of ligand-bound and ligand-free peaks. In this case, the positions of both peaks in the spectrum remained steady, and the changes occurred only in their intensities. The left panels in Figure 4A–D show the increase in the intensity of the bound-state Tyr 194 peak of CA I at a fixed pressure. Increasing ligand concentrations enhance the intensity of the peak corresponding to the ligand-bound state of CA I. Figure 3 shows the cross-peaks of CA I affected by compound 1 in the overlaid 1H– 15N HSQC spectrum at a pressure of 5 MPa. Many residues showed a second peak corresponding to the ligand-bound state upon addition of either compound 1 or 2. (39) In the calculations, we used the volume of amide cross-peaks in 1H– 15N HSQC spectra. The intensity ratio of ligand-free and ligand-bound peaks is proportional to the fraction of protein molar concentrations in each state. This exchange rate allowed the monitoring of peaks that correspond to ligand-free and ligand-bound amino acid residues of a protein in the same spectrum. (40,51) Two-dimensional 1H– 15N HSQC NMR spectra showed that both isoforms of carbonic anhydrase are in the slow exchange (in NMR time-scale) between two protein states-the ligand-bound and ligand-free-for compounds 1 and 2. In this study, we used two compounds that have a primary sulfonamide group in their structures ( Figure 2) and exhibit sub-millimolar binding affinities for CA I and CA II.
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(48) A full set of spectra for one sample throughout the pressure range was recorded in approximately 20 h with CA I and 30 h with CA II protein samples.
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All NMR experiments were processed with TOPSPIN software (Bruker), and the spectra were analyzed with CcpNmr Analysis V2 software. Water resonance was used as the reference because the most commonly used referencing compound sodium trimethylsilylpropanesulfonate (DSS) might inhibit CA I and CA II. (47) 1H chemical shifts were directly referenced to the water resonance (4.7 ppm), while 15N chemical shifts were referenced indirectly to the 15N/ 1H absolute frequency ratios. Water suppression was achieved using the WATERGATE method. 2D 1H– 15N HSQC spectra were recorded at 25 ☌ and eight different pressures ranging from 5 to 210 MPa on a Bruker AVANCE III 600 MHz equipped with a 5 mM Z-gradient TXI probe head. Hydrostatic pressure was applied to the sample directly within the magnet through an inox line filled with low-density paraffin oil (Sigma) using an Xtreme Syringe Pump from Daedalus Innovations. The protein solution (0.33 mL) was added into a ceramic tube with an outer diameter of 5 mM and an inner diameter of 3 mM from Daedalus Innovations (Aston, PA). High-pressure NMR spectroscopy was used to record 2D 1H– 15N HSQC spectra of CA I and CA II at various pressures. We think that the best way to obtain a complementary view of the protein–ligand binding volume is to use several techniques by exploiting their strengths and overcoming possible weaknesses. (38,39) All mentioned techniques reveal different aspects of protein–ligand interactions and have not only advantages but limitations also. (37) The protein–ligand dissociation constant, K d, can be accurately determined only if the protein concentration is in the range of K d, and thus the micromolar concentration of a protein in the NMR experiment limits the range of possible ligand affinities to weak and moderate.
Advantages of NMR spectroscopy come at a price: this assay requires relatively high concentrations of 15N-labeled proteins. Such features are unavailable in density- or fluorescence-based techniques, which provide ensemble-averaged properties, and many details remain hidden.
This allows identification of the binding-affected amino acid residues and analyze changes at the ligand binding site. (16,29,35,36) The NMR spectroscopy is particularly informative in volumetric measurements because it can monitor changes in the local amino acid rearrangement.
Various experimental approaches may be used to measure the protein–ligand binding volume including the density and ultrasound velocity techniques, (23,33) small-angle X-ray and elastic incoherent neutron scattering, (34) fluorescence spectroscopy at elevated pressures, (24,30,31) and high-pressure NMR.