Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Nuclear magnetic resonance, or NMR, spectroscopy enables atomic-level protein-protein interaction detection.

To study interactions between globular and helical proteins, label purified wild-type or mutant globular proteins with ¹⁵N — a nitrogen isotope. In an NMR tube, mix the labeled globular and unlabeled helical proteins in a solvent containing an NMR standard. Insert the tube into the NMR spectrometer.

The spectrometer magnets generate a strong magnetic field, aligning the ¹⁵N-labeled globular protein nuclei parallel to the field. A radiofrequency, or RF, pulse excites the ¹⁵N-nuclei to align antiparallel to the field.

The pulse frequency required for the transition of ¹⁵N nuclei between the two states defines the resonance frequency. Normalizing the ¹⁵N-resonance frequency to the NMR standard measures the ¹⁵N-chemical shift.

The magnetic field is adjusted to align the ¹H-nuclei — bonded to ¹⁵N — parallel to the field. The RF pulses are adjusted, and the ¹H-chemical shift is recorded.

During protein interactions, the chemical environment surrounding the interacting amino acid residues changes, altering the ¹⁵N-¹H chemical bonds and corresponding chemical shifts. Plot the ¹⁵N-¹H NMR spectra.

Without helical proteins, wild-type globular proteins exhibit well-resolved spectral peaks. When helical and wild-type globular proteins interact, the spectral peak is reduced. In contrast, incubating helical proteins with mutant globular proteins results in the spectral peaks reappearing, indicating no interaction between them.