Unfolding and pH studies on manganese peroxidase: Role of heme and calcium on secondary structure stability

Lucia Banci, Ilaria Bartalesi, Simone Ciofi-Baffoni, Ming Tien

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding- unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.

Original languageEnglish (US)
Pages (from-to)38-47
Number of pages10
JournalBiopolymers - Biospectroscopy Section
Volume72
Issue number1
DOIs
StatePublished - Jan 25 2003

Fingerprint

manganese peroxidase
Heme
Manganese
Calcium
Disulfides
Proteins
Ions
Dichroism
Reducing agents
Secondary Protein Structure
Prosthetics
Dithiothreitol
Reducing Agents
Circular Dichroism
Enzymes
Oxidants
Spectroscopy
Spectrum Analysis

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

Cite this

@article{3d5bf5ac934746a2826bc41ff7fb1b68,
title = "Unfolding and pH studies on manganese peroxidase: Role of heme and calcium on secondary structure stability",
abstract = "The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding- unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.",
author = "Lucia Banci and Ilaria Bartalesi and Simone Ciofi-Baffoni and Ming Tien",
year = "2003",
month = "1",
day = "25",
doi = "10.1002/bip.10283",
language = "English (US)",
volume = "72",
pages = "38--47",
journal = "Biopolymers",
issn = "0006-3525",
publisher = "John Wiley and Sons Inc.",
number = "1",

}

Unfolding and pH studies on manganese peroxidase : Role of heme and calcium on secondary structure stability. / Banci, Lucia; Bartalesi, Ilaria; Ciofi-Baffoni, Simone; Tien, Ming.

In: Biopolymers - Biospectroscopy Section, Vol. 72, No. 1, 25.01.2003, p. 38-47.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Unfolding and pH studies on manganese peroxidase

T2 - Role of heme and calcium on secondary structure stability

AU - Banci, Lucia

AU - Bartalesi, Ilaria

AU - Ciofi-Baffoni, Simone

AU - Tien, Ming

PY - 2003/1/25

Y1 - 2003/1/25

N2 - The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding- unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.

AB - The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding- unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.

UR - http://www.scopus.com/inward/record.url?scp=0037236592&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037236592&partnerID=8YFLogxK

U2 - 10.1002/bip.10283

DO - 10.1002/bip.10283

M3 - Article

C2 - 12400090

AN - SCOPUS:0037236592

VL - 72

SP - 38

EP - 47

JO - Biopolymers

JF - Biopolymers

SN - 0006-3525

IS - 1

ER -