Detection of intramolecular protein dynamics on nanosecond-to-microsecond timescales by nanoparticle-assisted NMR spin relaxation (NASR).

Proteins under physiological conditions have an intrinsically dynamic nature; they sample a multitude of different conformational substates that allow them to perform their biological functions. Protein motions can take place on a wide range of timescales. Although there are many different NMR experiments with sensitivity to different time windows, it has proven difficult to measure intramolecular motions that happen in the nanosecond-to-microsecond regime. Nanoparticle-assisted NMR spin relaxation (NASR) has recently been introduced to overcome this long-standing challenge. When colloidal nanoparticles are added to proteins in solution, the effective global tumbling of the protein molecules slows down, whereas the internal motions remain essentially unperturbed. NASR extends the protein dynamics observation window from picoseconds all the way into the microsecond range. In this protocol, the NASR effect is realized by using commercially available silica nanoparticles, and NMR measurements are acquired by using a standard high-field solution NMR spectrometer. NASR data analysis is shown to be straightforward. We demonstrate NASR by detecting sub-microsecond dynamics in the Switch I and II regions of oncogenic human KRAS and in the Loop I region of bacterial colicin-immunity protein Im7, among other protein constructs. When an isotope-labeled protein sample is available, this protocol can be executed in 2-5 d, including sample preparation, NMR experiments and data processing and analysis, to uncover potentially functionally important intramolecular dynamics at atomic resolution on timescales that are several orders of magnitude slower than what conventional spin relaxation experiments can observe.