NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes.
BACKGROUND
Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization.


RESULTS
Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening.


CONCLUSIONS
The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.
Maslennikov
Innokentiy
I
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037, USA. inno@salk.edu
Kefala
Georgia
G
Johnson
Casey
C
Riek
Roland
R
Choe
Senyon
S
Kwiatkowski
Witek
W
D002462:Cell Membrane / Q000737:chemistry; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results; D012995:Solubility; D014461:Ultracentrifugation
D003902:Detergents; D008565:Membrane Proteins