Abstract:
: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 Gα 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 Gα(i1) (hGα(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 hGα(i1) utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hGα(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 μs-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hGα(i1) (hGα(i1)-Δ31) 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 hGα(i1)-Δ31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/Gα binding selectivity.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Maly J,Crowhurst KAdoi
10.1016/j.pep.2012.06.003subject
Has Abstractpub_date
2012-08-01 00:00:00pages
255-64issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(12)00172-6journal_volume
84pub_type
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