Quantitative In-Cell Hydrogen NMR Spectroscopy to Monitor Protein-Ligand Interactions
Transcrição
To monitor the interactions of target proteins with small molecule test ligands in a native cellular environment, begin with an NMR tube filled with a buffer containing a gel plug at the base. Inject the target protein-expressing cells, embedded in agarose into the NMR tube, forming thread-like structures.
Load the tube into a bioreactor's flow unit, supplying a growth medium to maintain cell viability. Insert the flow unit inside a nuclear magnetic resonance or NMR instrument.
Under a strong magnetic field, the atomic nuclei of the proteins' hydrogen atoms get magnetized and align parallel to the field, adapting to a low-energy state.
Apply a radiofrequency pulse, causing nuclei to align anti-parallel to the magnetic field, allowing them to transition to higher-energy states.
After a brief pulse, the nuclei return to their original position, emitting absorbed energy. The detector captures this energy, generating an NMR spectrum representing the hydrogen atom's resonance frequencies. Each spectral peak corresponds to a different hydrogen atom in a specific electronic environment.
Add ligand molecules into the flow unit. These penetrate inside the cells, interact with the target protein, and alter the electronic environment around the hydrogen atoms, causing resonance frequency change.
Compare the NMR spectra. The spectral differences post-ligand binding indicate successful protein-ligand interaction.
