Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation.
Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.
Maslennikov
Innokentiy
I
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA.
Krupa
Martin
M
Dickson
Christopher
C
Esquivies
Luis
L
Blain
Katherine
K
Kefala
Georgia
G
Choe
Senyon
S
Kwiatkowski
Witek
W
D003902:Detergents / Q000737:chemistry*; D008027:Light; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D013499:Surface Properties; D014461:Ultracentrifugation / Q000379:methods*
D003902:Detergents; D008565:Membrane Proteins