title: Conformational states of HRPA1 induced by thermal unfolding: effect of low molecular weight solutes.
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.
authors.affiliation: Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais 1049-001, Lisboa, Portugal.
authors.forename: Ana Sofia L
authors.initials: AS
authors.lastname: Carvalho
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Santos
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Neves-Petersen
authors.forename: Steffen B
authors.initials: SB
authors.lastname: Petersen
authors.forename: Maria Raquel
authors.initials: MR
authors.lastname: Aires-Barros
authors.forename: Eduardo Pinho
authors.initials: EP
authors.lastname: e Melo
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D006358:Hot Temperature; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; C098053:HrpA1 protein, Xanthomonas campestris; D014364:Tryptophan