Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli

Abstract

A major protein of the outer membrane of Escherichia coli changed its apparent molecular weight upon heating in solutions containing sodium dodecyl sulfate. The difference between the unmodified form, B, and the heat-modified form, B ∗, in this detergent was investigated by a number of physical techniques. Protein B ∗ ( M r, 33,400) had a higher apparent molecular weight than form B ( M r, 28,500) as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulfate. Protein B ∗ had a lower sedimentation coefficient than form B, at all concentrations of detergents examined, indicating a more unfolded structure. This was supported by the more rapid digestion of protein B ∗ by Pronase and by its higher intrinsic viscosity. The partial specific volumes of the detergent-protein complexes of proteins B and B ∗ were similar at specified concentrations of sodium dodecyl sulfate. Ultraviolet difference spectroscopy and circular dichroism measurements indicated that protein B underwent conformational changes upon heating. These changes were not accompanied by a large increase in binding of sodium dodecyl sulfate as determined by equilibrium dialysis and from sedimentation and gel filtration data. Studies of protein B, freed of sodium dodecyl sulfate by ion exchange chromatography, showed that the detergent was not required for the conversion of protein B to form B ∗. We propose that protein B as extracted from the membrane contains some native structure which is lost upon heating.