Characterization of the refolding and reassembly of an integral membrane protein OmpF porin by low-angle laser light scattering photometry coupled with high-performance gel chromatography.
The refolding and reassembly of an integral membrane protein OmpF porin denatured in sodium dodecylsulfate (SDS) into its stable species by the addition of n-octyl-beta-D-glucopyranoside (OG) have been studied by means of circular dichroism (CD) spectroscopy and low-angle laser light scattering photometry coupled with high-performance gel chromatography. The minimal concentration where change in the secondary structure was induced by the addition of OG was found to be 6.0 mg/ml in CD experiments. A species unfolded further than the SDS-denatured form of this protein was observed at an early stage (5-15 min) of refolding just above the minimal OG concentration. In addition, the CD spectrum of protein species obtained above the minimal OG concentration showed that the protein is composed of a beta-structure which is different from the native structure of this protein. In light scattering experiments, no changes in molecular assemblies were observed when the OG concentration was below its minimal refolding concentration determined by CD measurements. Above the minimal concentration, a compact monomeric species was observed when denatured OmpF porin was incubated for 5 min at 25 degrees C in a refolding medium containing 1 mg/ml SDS and 7 mg/ml OG, and then injected into columns equilibrated with the refolding medium. After an incubation of 24 h before injection into the columns, predominant dimerization of this protein was observed in addition to incorrect aggregation.
Watanabe
Yasushi
Y
National Food Research Institute, Tsukuba, Ibaraki, Japan. yasuw@nfri.affrc.go.jp
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D010783:Photometry / Q000379:methods*; D018272:Porins / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013501:Surface-Active Agents / Q000737:chemistry
C109237:OmpF protein; D018272:Porins; D013501:Surface-Active Agents