Natural domain design

Enhanced thermal stability of a zinc-lacking ferredoxin isoform shows that a hydrophobic core efficiently replaces the structural metal site

Rita Rocha, Sónia S. Leal, Vitor H. Teixeira, Manuela Regalla, Harald Huber, António M. Baptista, Cláudio M. Soares, Cláudio M. Gomes

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11 Citations (Scopus)

Abstract

Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn 2+-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 ̊C. Surprisingly, the Zn 2+-lacking isoform was always found to be more stable than its Zn2+-containing counterpart: a ΔTm ≈ 9 °C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a ΔTm ≈ 24 °C at pH 2 and 10. The contribution of the Zn2+ site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the Tm in FdA (≈10 °C), whereas it has no effect in FdB. This shows that the Zn2+ site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.

Original languageEnglish
Pages (from-to)10376-10384
Number of pages9
JournalBiochemistry
Volume45
Issue number34
DOIs
Publication statusPublished - 29 Aug 2006

Fingerprint

Structural metals
Ferredoxins
Zinc
Protein Isoforms
Thermodynamic stability
Hot Temperature
Metals
Fluorescence
Sulfolobus
Protein Unfolding
Proteins
Structural Models
Molecular Dynamics Simulation
Chelating Agents
Tryptophan
Sequence Analysis
Molecular dynamics
Quenching
Availability
Ligands

Keywords

  • Quenching
  • Structural metals
  • Absorption
  • Fluorescence
  • Hydrophobicity
  • pH

Cite this

@article{719a8f8cccdd49c19e9783a23d2046f5,
title = "Natural domain design: Enhanced thermal stability of a zinc-lacking ferredoxin isoform shows that a hydrophobic core efficiently replaces the structural metal site",
abstract = "Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn 2+-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 ̊C. Surprisingly, the Zn 2+-lacking isoform was always found to be more stable than its Zn2+-containing counterpart: a ΔTm ≈ 9 °C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a ΔTm ≈ 24 °C at pH 2 and 10. The contribution of the Zn2+ site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the Tm in FdA (≈10 °C), whereas it has no effect in FdB. This shows that the Zn2+ site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.",
keywords = "Quenching, Structural metals, Absorption, Fluorescence, Hydrophobicity, pH",
author = "Rita Rocha and Leal, {S{\'o}nia S.} and Teixeira, {Vitor H.} and Manuela Regalla and Harald Huber and Baptista, {Ant{\'o}nio M.} and Soares, {Cl{\'a}udio M.} and Gomes, {Cl{\'a}udio M.}",
year = "2006",
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TY - JOUR

T1 - Natural domain design

T2 - Enhanced thermal stability of a zinc-lacking ferredoxin isoform shows that a hydrophobic core efficiently replaces the structural metal site

AU - Rocha, Rita

AU - Leal, Sónia S.

AU - Teixeira, Vitor H.

AU - Regalla, Manuela

AU - Huber, Harald

AU - Baptista, António M.

AU - Soares, Cláudio M.

AU - Gomes, Cláudio M.

PY - 2006/8/29

Y1 - 2006/8/29

N2 - Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn 2+-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 ̊C. Surprisingly, the Zn 2+-lacking isoform was always found to be more stable than its Zn2+-containing counterpart: a ΔTm ≈ 9 °C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a ΔTm ≈ 24 °C at pH 2 and 10. The contribution of the Zn2+ site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the Tm in FdA (≈10 °C), whereas it has no effect in FdB. This shows that the Zn2+ site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.

AB - Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn 2+-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 ̊C. Surprisingly, the Zn 2+-lacking isoform was always found to be more stable than its Zn2+-containing counterpart: a ΔTm ≈ 9 °C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a ΔTm ≈ 24 °C at pH 2 and 10. The contribution of the Zn2+ site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the Tm in FdA (≈10 °C), whereas it has no effect in FdB. This shows that the Zn2+ site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.

KW - Quenching

KW - Structural metals

KW - Absorption

KW - Fluorescence

KW - Hydrophobicity

KW - pH

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U2 - 10.1021/bi0610698

DO - 10.1021/bi0610698

M3 - Article

VL - 45

SP - 10376

EP - 10384

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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