Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1

Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigation of intramolecular motions in RGS and G alpha proteins in their apo and complexed forms, we have successfully developed a protocol for preparing milligram quantities of highly purified, isotopically labeled wild-type human G alpha(i1) (hG alpha(i1)) subunit for NMR studies. High levels of expression in Escherichia coli can be attributed to the use of the SUMO fusion protein system, a bacterial strain that produces rare codons, supplementation of minimal medium with small quantities of isotopically labeled rich medium and a lowered induction temperature. Purification of hG alpha(i1) utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hG alpha(i1) has shown that high-quality spectra can be obtained at near-physiological temperatures, whereas lower temperature spectra display numerous weak and broadened peaks, providing preliminary evidence for widespread mu s-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hG alpha(i1) (hG alpha(i1)-Delta 31) in which the 31-residue unstructured N-terminus was removed. This resulted in further improvements in spectral quality by eliminating high-intensity peaks that obscured resonances from structured segments of the protein. We plan to use hG alpha(i1)-Delta 31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/G alpha binding selectivity. (C) 2012 Elsevier Inc. All rights reserved.