title: Formation of a misfolded conformation during refolding of HRPA1 in the presence of calcium.
abstract: Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca2+-depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, ICa2+, is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence. The fraction of ICa2+ increases exponentially with increasing calcium concentration. The ICa2+ state is formed during refolding after calcium binding to the unfolded state, as reconstitution of HRPA1 from its apoprotein reveals that the affinity of the apoprotein to protoporphyrin IX is higher in the presence of calcium. If calcium is added after refolding only, the majority of HRPA1 molecules retain their native conformation, thus confirming the binding of calcium to the unfolded state.
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: 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
authors.initials: E
authors.lastname: Pinho e Melo
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D011524:Protoporphyrins / Q000737:chemistry; D013696:Temperature
chemical_list: D011524:Protoporphyrins; D006418:Heme; C028025:protoporphyrin IX; D006735:Horseradish Peroxidase; D002118:Calcium