期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 84, 期 2, 页码 255-264出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.06.003
关键词
G protein alpha subunit; Signaling protein; Protein NMR; SUMO fusion; Isotope labeling
类别
资金
- NSF [MCB-1158177]
- College of Science and Mathematics
- Office of Sponsored Projects at California State University, Northridge
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1158177] Funding Source: National Science Foundation
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.
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