HTRA HEAT-SHOCK PROTEASE INTERACTS WITH PHOSPHOLIPID-MEMBRANES AND UNDERGOES CONFORMATIONAL-CHANGES

The HtrA (DegP) protein of Escherichia coli is a heat shock serine pro tease, essential for cell survival only at temperatures above 42 degre es C. It has been shown by genetic experiments that HtrA is an envelop e protease, functioning in the periplasmic space. To clarify the cellu lar localization of HtrA, E. coli cells were fractionated, and HtrA wa s not detected by the immunoblotting technique in the periplasm or in the fraction of soluble proteins but was found in the inner membrane. The protein could be partially eluted from the total membrane fraction by a high ionic strength solution, whereas solutions affecting protei n conformation released HtrA almost com pletely. These results, taken together with the evidence showing that HtrA functions in the periplas m, indicate that HtrA is a peripheral membrane protein, localized on t he periplasmic side of the inner membrane. As the first step toward so lving the problem of HtrA-membrane interactions, the structure of HtrA in the presence of phosphatidylglycerol (PG), phosphatidylethanolamin e (PE), or cardiolipin (CL) was analyzed by fluorescence and Fourier-t ransform infrared spectroscopy. The infrared and fluorescence data ind icated an interaction of HtrA with PG and CL but not with PE suspensio ns. Fluorescence spectroscopy revealed that this interaction was at th e level of the polar head group of the phospholipid. In the PG/HtrA sy stem, small changes were observed in the HtrA secondary structure and a remarkable decrease of the thermal stability of the protein, which s uggested changes in HtrA tertiary structure. This suggestion was suppo rted by fluorescence data that showed a shift of the fluorescence emis sion spectrum of HtrA tyrosine residues in the presence of PG and a re duced fluorescence intensity, phenomena not observed in the presence o f PE or CL suspensions. Infrared data revealed also that the interacti on of HtrA with PG leads to a protection of unfolded protein against a ggregation at relatively low temperatures. The conformational changes of HtrA in the presence of PG influenced the proteolytic activity of H trA by increasing it at the temperatures 37-45 degrees C and inhibitin g it at 50-55 degrees C. CL inhibited HtrA activity at all of the temp eratures tested.