Recently, a research team led by Prof. WANG Junfeng from the High Magnetic Field Laboratory, Hefei Institutes of Physical Science (HFIPS) of Chinese Academy of Sciences (CAS), developed a new 1D 19F NMR-based method for rapid detection of Arf6 guanine nucleotide exchange factor (GEF) activity.
The results were recently published in Analytical Chemistry.
Small GTPases (sGTPase) are involved in signal cascades that control a wide range of cellular responses. All sGTPases can be found in two states: An inactive state in which the small GTPase is bound to GDP, and an active state in which it is bound to GTP. The GTPase cycle formed by the interconversion between the inactive and activated states is strictly regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).
Mutation or abnormal regulation of sGTPase proteins or their respective GEFs/GAPs are closely related to many of diseases including cancer. Therefore, it is very important to detect the activity of these regulatory GEF/GAP proteins both in vitro and in vivo.
In this research, the researchers incorporated the unnatural amino acid tfmF site-specifically into Phe47 of sGTPase Arf6, a position located in the ARF6 switching domain whose conformation changes most during GTPase cycle. A fast and robust 1D 19F NMR-based method was developed to detect the GEF activity in purified protein system or in the presence of the cell lysates.
For a GEF activity assay, the sample of GDP loaded Arf6-F47 tfmF was put into the spectrometer as soon as possible after adding a GEF and GTP. A sequential series of 1D 19F NMR spectra were collected. The signal of Arf6-F47 tfmF in the GTP-bound state was processed and integrated. The exchange kinetic curves were then plotted and fitted to a single-phase exponential growth curve to extract the rate.
"This strategy could potentially be used to monitor the conformational change of Arf6 or other sGTPase," said WU Bo, first author of the paper, "it could detect the activities of sGTPases regulatory proteins in physiology and pathology environments."
This research was supported by the National Natural Science Foundation of China (21673244), the Collaborative Innovation Program of Hefei Science Center, CAS (2020HSC-CIP008), and the High Magnetic Field Laboratory of Anhui Province. All the NMR experiments were performed at the High Magnetic Field Laboratory, Chinese Academy of Sciences.
Figure: 19F NMR-based method for Arf6 GEF activity detection (Image by WU Bo)