Conformational states of HRPA1 induced by thermal unfolding: effect of low molecular weight solutes |
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Authors: | Carvalho Ana Sofia L Santos Andrea M Neves-Petersen Maria Teresa Petersen Steffen B Aires-Barros Maria Raquel e Melo Eduardo Pinho |
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Institution: | Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais 1049-001, Lisboa, Portugal. |
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Abstract: | Fluorescence, CD, and activity measurements were used to characterize the different conformational states of horseradish peroxidase A1 induced by thermal unfolding. Picosecond time-resolved fluorescence studies showed a three-exponential decay dominated by a picosecond lifetime component resulting from energy transfer from tryptophan to heme. Upon thermal unfolding a decrease in the preexponential factor of the picosecond lifetime and an increase in the quantum yield were observed approaching the characteristics observed for apoHRPA1. The fraction of heme-quenched fluorophore decreased to 0.4 after unfolding as shown by acrylamide quenching. A new unfolding pathway for HRPA1 was proposed and the effect of the low molecular weight solutes trehalose, sorbitol, and melezitose on this pathway was analyzed. Native HRPA1 unfolds with an intermediate between the native and the unfolded conformation. The unfolded conformation can refold to the native state or to a native-like conformation with no calcium ions upon cooling or can give an irreversible denatured state. The refolded conformation with no calcium ions was clearly identified in a second thermal scan in the presence of EDTA and shows secondary and tertiary structures, heme reincorporation in the cavity, and at least 59% of activity. Melezitose stabilized the refolded Ca2+-depleted protein and induced a more complex mechanism for heme disruption. The effect of sorbitol and trehalose were mainly characterized by an increase in the temperature of unfolding. |
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Keywords: | horseradish peroxidase A1 unfolding pathway tryptophan fluorescence lifetimes acrylamide quenching CD low molecular weight solutes peroxidase calcium ions unfolding intermediate state heme fluorescence resonance energy transfer |
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