Protein folding intermediates of invasin protein IbeA from Escherichia coli

Damodara R. Mendu, Venkata R. Dasari, Mian Cai, Kwang S. Kim

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His6-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of β-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with β-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended β-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.

Original languageEnglish (US)
Pages (from-to)458-469
Number of pages12
JournalFEBS Journal
Volume275
Issue number3
DOIs
StatePublished - Feb 2008
Externally publishedYes

Fingerprint

Protein folding
Escherichia coli Proteins
Protein Folding
Escherichia coli
Sulfonic Acids
Molten materials
His-His-His-His-His-His
Guanidine
Proteins
Acids
Fusion reactions
Fluorescence
Denaturation
Endothelial cells
Tryptophan
Molecular structure
Conformations
Quenching
Brain
Molecular Structure

Keywords

  • Acid and Gdm-HCl-induced unfolding
  • Escherichia coli
  • Molten globule
  • Protein unfolding intermediates of IbeA

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Protein folding intermediates of invasin protein IbeA from Escherichia coli. / Mendu, Damodara R.; Dasari, Venkata R.; Cai, Mian; Kim, Kwang S.

In: FEBS Journal, Vol. 275, No. 3, 02.2008, p. 458-469.

Research output: Contribution to journalArticle

Mendu, Damodara R. ; Dasari, Venkata R. ; Cai, Mian ; Kim, Kwang S. / Protein folding intermediates of invasin protein IbeA from Escherichia coli. In: FEBS Journal. 2008 ; Vol. 275, No. 3. pp. 458-469.
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N2 - IbeA of Escherichia coli K1 was cloned, expressed and purified as a His6-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of β-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with β-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended β-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.

AB - IbeA of Escherichia coli K1 was cloned, expressed and purified as a His6-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of β-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with β-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended β-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.

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