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 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.
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA 91330-8262, USA.
Jan
J
Maly
Karin A
KA
Crowhurst
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000302:isolation & purification
D044385:GTP-Binding Protein alpha Subunits; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D006153:Guanosine Diphosphate; D008274:Magnesium