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
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Abstract
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.