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(Received for publication, July 22, 1996, and in revised form, January 8, 1997)
From the The HtrA (DegP) protein of
Escherichia coli is a heat shock serine protease, essential
for cell survival only at temperatures above 42 °C. It has been
shown by genetic experiments that HtrA is an envelope protease,
functioning in the periplasmic space. To clarify the cellular
localization of HtrA, E. coli cells were fractionated, and
HtrA was 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
protein conformation released HtrA almost completely. These results,
taken together with the evidence showing that HtrA functions in the
periplasm, indicate that HtrA is a peripheral membrane protein,
localized on the periplasmic side of the inner membrane. As the first
step toward solving the problem of HtrA-membrane interactions, the
structure of HtrA in the presence of phosphatidylglycerol (PG),
phosphatidylethanolamine (PE), or cardiolipin (CL) was analyzed by
fluorescence and Fourier-transform infrared spectroscopy. The infrared
and fluorescence data indicated an interaction of HtrA with PG and CL
but not with PE suspensions. Fluorescence spectroscopy revealed that
this interaction was at the level of the polar head group of the
phospholipid. In the PG/HtrA system, small changes were observed in the
HtrA secondary structure and a remarkable decrease of the thermal
stability of the protein, which suggested changes in HtrA tertiary
structure. This suggestion was supported by fluorescence data that
showed a shift of the fluorescence emission spectrum of HtrA tyrosine residues in the presence of PG and a reduced fluorescence intensity, phenomena not observed in the presence of PE or CL suspensions. Infrared data revealed also that the interaction of HtrA with PG leads
to a protection of unfolded protein against aggregation at relatively
low temperatures. The conformational changes of HtrA in the presence of
PG influenced the proteolytic activity of HtrA by increasing it at the
temperatures 37-45 °C and inhibiting it at 50-55 °C. CL
inhibited HtrA activity at all of the temperatures tested.
Volume 272, Number 14,
Issue of April 4, 1997
pp. 8974-8982
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
ska
,
,
Department of Biochemistry, University of
Gdask, K
adki 24, 80-822 Gdask, Poland and
¶ Institute of Biochemistry, Medical School, University of Ancona,
60131 Ancona, Italy
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