Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example.
For structural studies of integral membrane proteins, including their 3D crystallization, the judicious use of detergent for solubilization and purification is required. Detergent binding by the solubilized protein is an important parameter to determine the hydrodynamic properties in terms of size and aggregational (monomeric/oligo(proto)meric) state of the protein. Detergent binding can be measured by gel filtration chromatography under equilibrium conditions and after separation from mixed micelles of solubilized lipid and detergent. Using sarcoplasmic reticulum Ca(2+)-ATPase as an example, we demonstrate in this protocol complete procedures for measurement of detergent binding using (i) radiolabeled n-dodecyl-beta-D-maltoside (DM) or (ii) from measurements of the increase in refractive index due to the presence of bound detergent on the protein. The latter measurement can also be performed by sedimentation velocity (SV) analysis in the analytical ultracentrifuge which in addition allows determination of the sedimentation coefficient. In combination with estimation of Stokes radius by gel filtration calibration, the molecular mass and asymmetry of the solubilized protein can be calculated. In the proposed protocols, the gel chromatographic procedures require 1 d; SV experiments are performed just after size exclusion. The whole time for these experiments is 24 h. Data analysis of analytical ultracentrifugation requires a couple of days.
le Maire
Marc
M
CEA, Institut de Biologie et Technologies de Saclay, F-91191 Gif-sur-Yvette, France. marc.lemaire@cea.fr
Arnou
Bertrand
B
Olesen
Claus
C
Georgin
Dominique
D
Ebel
Christine
C
Møller
Jesper V
JV
D000818:Animals; D002138:Calibration; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D014461:Ultracentrifugation / Q000379:methods
D003902:Detergents; D008565:Membrane Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases