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Default Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein ?i1

Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein ?i1

August 2012
Publication year: 2012
Source:Protein Expression and Purification, Volume 84, Issue 2



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? 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?i1 (hG?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?i1 utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hG?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 ?s-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hG?i1 (hG?i1-?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?i1-?31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/G? binding selectivity.
Highlights

? A protocol was developed to prepare pure isotopically labeled human G?i1 for NMR. ? N-terminally truncated G?i1 (with unstructured residues removed) was also prepared. ? Success depended on vector choice, low induction temperature and media supplements. ? NMR spectra recorded at 35°C had better peak definition than at lower temperatures. ? Spectra provided preliminary evidence for widespread intramolecular motions in G?i1.





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