Insights into signal transduction by a hybrid FixL

Denaturation study of on and off states of a multi-domain oxygen sensor

Wellinson G. Guimarães, Ana C.S. Gondim, Pedro Mikael da Silva Costa, Marie Alda Gilles-Gonzalez, Luiz G.F. Lopes, Marta S.P. Carepo, Eduardo H.S. Sousa

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN) form was monitored by UV–visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV–visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8 kJ mol− 1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2 kJ mol− 1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.

Original languageEnglish (US)
Pages (from-to)129-137
Number of pages9
JournalJournal of Inorganic Biochemistry
Volume172
DOIs
StatePublished - Jul 1 2017

Fingerprint

Oxygen sensors
Signal transduction
Denaturation
Heme
Signal Transduction
Circular Dichroism
Oxygen
Spectrum Analysis
Fluorescence Spectrometry
Fluorescence spectroscopy
Cyanides
Dichroism
Absorption spectroscopy
Urea
Rhizobium etli
Phosphotransferases
Iron
Hybrid sensors
Circular dichroism spectroscopy
Hydrogen Bonding

Keywords

  • Conformational changes
  • FixL
  • Heme-based sensor
  • Oxygen sensor
  • Stability studies
  • Urea

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

Insights into signal transduction by a hybrid FixL : Denaturation study of on and off states of a multi-domain oxygen sensor. / Guimarães, Wellinson G.; Gondim, Ana C.S.; Costa, Pedro Mikael da Silva; Gilles-Gonzalez, Marie Alda; Lopes, Luiz G.F.; Carepo, Marta S.P.; Sousa, Eduardo H.S.

In: Journal of Inorganic Biochemistry, Vol. 172, 01.07.2017, p. 129-137.

Research output: Contribution to journalArticle

Guimarães, Wellinson G. ; Gondim, Ana C.S. ; Costa, Pedro Mikael da Silva ; Gilles-Gonzalez, Marie Alda ; Lopes, Luiz G.F. ; Carepo, Marta S.P. ; Sousa, Eduardo H.S. / Insights into signal transduction by a hybrid FixL : Denaturation study of on and off states of a multi-domain oxygen sensor. In: Journal of Inorganic Biochemistry. 2017 ; Vol. 172. pp. 129-137.
@article{84412e08afd947e8bf5109a1398ad258,
title = "Insights into signal transduction by a hybrid FixL: Denaturation study of on and off states of a multi-domain oxygen sensor",
abstract = "FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN−) form was monitored by UV–visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV–visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8 kJ mol− 1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2 kJ mol− 1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.",
keywords = "Conformational changes, FixL, Heme-based sensor, Oxygen sensor, Stability studies, Urea",
author = "Guimar{\~a}es, {Wellinson G.} and Gondim, {Ana C.S.} and Costa, {Pedro Mikael da Silva} and Gilles-Gonzalez, {Marie Alda} and Lopes, {Luiz G.F.} and Carepo, {Marta S.P.} and Sousa, {Eduardo H.S.}",
year = "2017",
month = "7",
day = "1",
doi = "10.1016/j.jinorgbio.2017.04.013",
language = "English (US)",
volume = "172",
pages = "129--137",
journal = "Journal of Inorganic Biochemistry",
issn = "0162-0134",
publisher = "Elsevier Inc.",

}

TY - JOUR

T1 - Insights into signal transduction by a hybrid FixL

T2 - Denaturation study of on and off states of a multi-domain oxygen sensor

AU - Guimarães, Wellinson G.

AU - Gondim, Ana C.S.

AU - Costa, Pedro Mikael da Silva

AU - Gilles-Gonzalez, Marie Alda

AU - Lopes, Luiz G.F.

AU - Carepo, Marta S.P.

AU - Sousa, Eduardo H.S.

PY - 2017/7/1

Y1 - 2017/7/1

N2 - FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN−) form was monitored by UV–visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV–visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8 kJ mol− 1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2 kJ mol− 1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.

AB - FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN−) form was monitored by UV–visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV–visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8 kJ mol− 1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2 kJ mol− 1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.

KW - Conformational changes

KW - FixL

KW - Heme-based sensor

KW - Oxygen sensor

KW - Stability studies

KW - Urea

UR - http://www.scopus.com/inward/record.url?scp=85018185960&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85018185960&partnerID=8YFLogxK

U2 - 10.1016/j.jinorgbio.2017.04.013

DO - 10.1016/j.jinorgbio.2017.04.013

M3 - Article

VL - 172

SP - 129

EP - 137

JO - Journal of Inorganic Biochemistry

JF - Journal of Inorganic Biochemistry

SN - 0162-0134

ER -