Temperature-induced conformational changes within the regulatory loops L1–L2–LA of the HtrA heat-shock protease from Escherichia coli

Abstract

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25–45 °C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes ( τ) and wavelength emission maxima ( λ emmax) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25–30 °C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 °C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 °C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.