title: 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 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) 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 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.
authors.affiliation: Department of Biochemistry, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Sobiecka-Szkatula
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Polit
authors.forename: Andrea
authors.initials: A
authors.lastname: Scire
authors.forename: Artur
authors.initials: A
authors.lastname: Gieldon
authors.forename: Fabio
authors.initials: F
authors.lastname: Tanfani
authors.forename: Zaneta
authors.initials: Z
authors.lastname: Szkarlat
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ciarkowski
authors.forename: Dorota
authors.initials: D
authors.lastname: Zurawa-Janicka
authors.forename: Joanna
authors.initials: J
authors.lastname: Skorko-Glonek
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipinska
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response / Q000235:genetics; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; C061682:DegP protease; D012697:Serine Endopeptidases