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Kinetics of salt-dependent unfolding of [2Fe–2S] ferredoxin of Halobacterium salinarum

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Abstract

The [2Fe–2S] ferredoxin from the extreme haloarchaeon Halobacterium salinarum is stable in high (>1.5 M) salt concentration. At low salt concentration the protein exhibits partial unfolding. The kinetics of unfolding was studied in low salt and in presence of urea in order to investigate the role of salt ions on the stability of the protein. The urea dependent unfolding, monitored by fluorescence of the tryptophan residues and circular dichroism, suggests that the native protein is stable at neutral pH, is destabilized in both acidic and alkaline environment, and involves the formation of kinetic intermediate(s). In contrast, the unfolding kinetics in low salt exhibits enhanced rate of unfolding with increase in pH value and is a two state process without the formation of intermediate. The unfolding at neutral pH is salt concentration dependent and occurs in two stages. The first stage, involves an initial fast phase (indicative of the formation of a hydrophobic collapsed state) followed by a relatively slow phase, and is dependent on the type of cation and anion. The second stage is considerably slower, proceeds with an increase in fluorescence intensity and is largely independent of the nature of salt. Our results thus show that the native form of the haloarchaeal ferredoxin (in high salt concentration) unfolds in low salt concentration through an apparently hydrophobic collapsed form, which leads to a kinetic intermediate. This intermediate then unfolds further to the low salt form of the protein.

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Abbreviations

CAPSO:

3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid

CD:

Circular dichroism

FL:

Fluorescence

HmMDH:

Halobacterium marismortui malate dehydrogenase

HmFd:

Halobacterium marismortui ferredoxin

HsFd:

Halobacterium salinarum ferredoxin

MES:

2-morpholinoethanesulfonic acid

MOPS:

3-(N-morpholino)propanesulfonic acid

NATA:

N-acetyltryptophanamide

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Acknowledgments

We are grateful to Prof. A. K. Singh, Department of Chemistry, Indian Institute of Technology, Mumbai for providing the H. salinarum strain M1.

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Author notes

  1. Amal K. Bandyopadhyay

    Present address: Department of Biotechnology, University of Burdwan, West Bengal, India

Authors and Affiliations

  1. Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai , 400 005, India

    Amal K. Bandyopadhyay, G. Krishnamoorthy & Haripalsingh M. Sonawat

  2. Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400 005, India

    Lakshmi C. Padhy

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  1. Amal K. Bandyopadhyay
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  2. G. Krishnamoorthy
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Correspondence to Haripalsingh M. Sonawat.

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Communicated by K. Horikoshi.

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Bandyopadhyay, A.K., Krishnamoorthy, G., Padhy, L.C. et al. Kinetics of salt-dependent unfolding of [2Fe–2S] ferredoxin of Halobacterium salinarum . Extremophiles 11, 615–625 (2007). https://doi.org/10.1007/s00792-007-0075-0

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