Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein.
Identifieur interne : 003229 ( Main/Merge ); précédent : 003228; suivant : 003230Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein.
Auteurs : Mehmet A. Kilic [Royaume-Uni] ; Stephen Spiro ; Geoffrey R. MooreSource :
- Protein science : a publication of the Protein Society [ 0961-8368 ] ; 2003.
Descripteurs français
- KwdFr :
- Biopolymères (), Biopolymères (génétique), Biopolymères (métabolisme), Concentration en ions d'hydrogène, Conformation des protéines (), Cytochromes de type b (), Cytochromes de type b (génétique), Cytochromes de type b (métabolisme), Dichroïsme circulaire, Dénaturation des protéines (), Ferritines (), Ferritines (génétique), Ferritines (métabolisme), Modèles moléculaires, Mutagenèse dirigée, Mutation (génétique), Pliage des protéines, Protéines bactériennes (), Protéines bactériennes (génétique), Protéines bactériennes (métabolisme), Relation structure-activité, Rhodobacter capsulatus (), Rhodobacter capsulatus (génétique), Température, Thermodynamique, Urée (pharmacologie), Électrophorèse sur gel d'agar.
- MESH :
- génétique : Biopolymères, Cytochromes de type b, Ferritines, Mutation, Protéines bactériennes, Rhodobacter capsulatus.
- métabolisme : Biopolymères, Cytochromes de type b, Ferritines, Protéines bactériennes.
- pharmacologie : Urée.
- Biopolymères, Concentration en ions d'hydrogène, Conformation des protéines, Cytochromes de type b, Dichroïsme circulaire, Dénaturation des protéines, Ferritines, Modèles moléculaires, Mutagenèse dirigée, Pliage des protéines, Protéines bactériennes, Relation structure-activité, Rhodobacter capsulatus, Température, Thermodynamique, Électrophorèse sur gel d'agar.
English descriptors
- KwdEn :
- Bacterial Proteins (chemistry), Bacterial Proteins (genetics), Bacterial Proteins (metabolism), Biopolymers (chemistry), Biopolymers (genetics), Biopolymers (metabolism), Circular Dichroism, Cytochrome b Group (chemistry), Cytochrome b Group (genetics), Cytochrome b Group (metabolism), Electrophoresis, Agar Gel, Ferritins (chemistry), Ferritins (genetics), Ferritins (metabolism), Hydrogen-Ion Concentration, Models, Molecular, Mutagenesis, Site-Directed, Mutation (genetics), Protein Conformation (drug effects), Protein Denaturation (drug effects), Protein Folding, Rhodobacter capsulatus (chemistry), Rhodobacter capsulatus (genetics), Structure-Activity Relationship, Temperature, Thermodynamics, Urea (pharmacology).
- MESH :
- chemical , chemistry : Bacterial Proteins, Biopolymers, Cytochrome b Group, Ferritins.
- chemical , genetics : Bacterial Proteins, Biopolymers, Cytochrome b Group, Ferritins.
- chemical , metabolism : Bacterial Proteins, Biopolymers, Cytochrome b Group, Ferritins.
- chemistry : Rhodobacter capsulatus.
- drug effects : Protein Conformation, Protein Denaturation.
- genetics : Mutation, Rhodobacter capsulatus.
- chemical , pharmacology : Urea.
- Circular Dichroism, Electrophoresis, Agar Gel, Hydrogen-Ion Concentration, Models, Molecular, Mutagenesis, Site-Directed, Protein Folding, Structure-Activity Relationship, Temperature, Thermodynamics.
Abstract
The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.
DOI: 10.1110/ps.0301903
PubMed: 12876316
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pubmed:12876316Le document en format XML
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<term>Bacterial Proteins (genetics)</term>
<term>Bacterial Proteins (metabolism)</term>
<term>Biopolymers (chemistry)</term>
<term>Biopolymers (genetics)</term>
<term>Biopolymers (metabolism)</term>
<term>Circular Dichroism</term>
<term>Cytochrome b Group (chemistry)</term>
<term>Cytochrome b Group (genetics)</term>
<term>Cytochrome b Group (metabolism)</term>
<term>Electrophoresis, Agar Gel</term>
<term>Ferritins (chemistry)</term>
<term>Ferritins (genetics)</term>
<term>Ferritins (metabolism)</term>
<term>Hydrogen-Ion Concentration</term>
<term>Models, Molecular</term>
<term>Mutagenesis, Site-Directed</term>
<term>Mutation (genetics)</term>
<term>Protein Conformation (drug effects)</term>
<term>Protein Denaturation (drug effects)</term>
<term>Protein Folding</term>
<term>Rhodobacter capsulatus (chemistry)</term>
<term>Rhodobacter capsulatus (genetics)</term>
<term>Structure-Activity Relationship</term>
<term>Temperature</term>
<term>Thermodynamics</term>
<term>Urea (pharmacology)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Biopolymères ()</term>
<term>Biopolymères (génétique)</term>
<term>Biopolymères (métabolisme)</term>
<term>Concentration en ions d'hydrogène</term>
<term>Conformation des protéines ()</term>
<term>Cytochromes de type b ()</term>
<term>Cytochromes de type b (génétique)</term>
<term>Cytochromes de type b (métabolisme)</term>
<term>Dichroïsme circulaire</term>
<term>Dénaturation des protéines ()</term>
<term>Ferritines ()</term>
<term>Ferritines (génétique)</term>
<term>Ferritines (métabolisme)</term>
<term>Modèles moléculaires</term>
<term>Mutagenèse dirigée</term>
<term>Mutation (génétique)</term>
<term>Pliage des protéines</term>
<term>Protéines bactériennes ()</term>
<term>Protéines bactériennes (génétique)</term>
<term>Protéines bactériennes (métabolisme)</term>
<term>Relation structure-activité</term>
<term>Rhodobacter capsulatus ()</term>
<term>Rhodobacter capsulatus (génétique)</term>
<term>Température</term>
<term>Thermodynamique</term>
<term>Urée (pharmacologie)</term>
<term>Électrophorèse sur gel d'agar</term>
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<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Bacterial Proteins</term>
<term>Biopolymers</term>
<term>Cytochrome b Group</term>
<term>Ferritins</term>
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<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Bacterial Proteins</term>
<term>Biopolymers</term>
<term>Cytochrome b Group</term>
<term>Ferritins</term>
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<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Bacterial Proteins</term>
<term>Biopolymers</term>
<term>Cytochrome b Group</term>
<term>Ferritins</term>
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<keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Protein Conformation</term>
<term>Protein Denaturation</term>
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<term>Rhodobacter capsulatus</term>
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<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Biopolymères</term>
<term>Cytochromes de type b</term>
<term>Ferritines</term>
<term>Mutation</term>
<term>Protéines bactériennes</term>
<term>Rhodobacter capsulatus</term>
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<term>Cytochromes de type b</term>
<term>Ferritines</term>
<term>Protéines bactériennes</term>
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<keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Urée</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Urea</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Circular Dichroism</term>
<term>Electrophoresis, Agar Gel</term>
<term>Hydrogen-Ion Concentration</term>
<term>Models, Molecular</term>
<term>Mutagenesis, Site-Directed</term>
<term>Protein Folding</term>
<term>Structure-Activity Relationship</term>
<term>Temperature</term>
<term>Thermodynamics</term>
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<keywords scheme="MESH" xml:lang="fr"><term>Biopolymères</term>
<term>Concentration en ions d'hydrogène</term>
<term>Conformation des protéines</term>
<term>Cytochromes de type b</term>
<term>Dichroïsme circulaire</term>
<term>Dénaturation des protéines</term>
<term>Ferritines</term>
<term>Modèles moléculaires</term>
<term>Mutagenèse dirigée</term>
<term>Pliage des protéines</term>
<term>Protéines bactériennes</term>
<term>Relation structure-activité</term>
<term>Rhodobacter capsulatus</term>
<term>Température</term>
<term>Thermodynamique</term>
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<front><div type="abstract" xml:lang="en">The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.</div>
</front>
</TEI>
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