Encyclopedia of Experiments: Biological Techniques
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Transcript
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.
