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Protein folding intermediates of invasin protein IbeA from Escherichia coli.
FEBS J. 2008 Feb; 275(3):458-69.FJ

Abstract

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-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 beta-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 beta-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 beta-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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Publisher Full Text (DOI)

Authors+Show Affiliations

Mendu DR
Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Dasari VR
No affiliation info available
Cai M
No affiliation info available
Kim KS
No affiliation info available

MeSH

Blotting, WesternBrainCells, CulturedCircular DichroismEndothelial CellsEscherichia coliEscherichia coli ProteinsHumansMembrane ProteinsMicroscopy, FluorescenceProtein FoldingProtons

Pub Type(s)

Journal Article

Language

eng

PubMed ID

18167139

Citation

Mendu, Damodara R., et al. "Protein Folding Intermediates of Invasin Protein IbeA From Escherichia Coli." The FEBS Journal, vol. 275, no. 3, 2008, pp. 458-69.
Mendu DR, Dasari VR, Cai M, et al. Protein folding intermediates of invasin protein IbeA from Escherichia coli. FEBS J. 2008;275(3):458-69.
Mendu, D. R., Dasari, V. R., Cai, M., & Kim, K. S. (2008). Protein folding intermediates of invasin protein IbeA from Escherichia coli. The FEBS Journal, 275(3), 458-69. https://doi.org/10.1111/j.1742-4658.2007.06213.x
Mendu DR, et al. Protein Folding Intermediates of Invasin Protein IbeA From Escherichia Coli. FEBS J. 2008;275(3):458-69. PubMed PMID: 18167139.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Protein folding intermediates of invasin protein IbeA from Escherichia coli. AU - Mendu,Damodara R, AU - Dasari,Venkata R, AU - Cai,Mian, AU - Kim,Kwang S, Y1 - 2007/12/20/ PY - 2008/1/3/pubmed PY - 2008/6/18/medline PY - 2008/1/3/entrez SP - 458 EP - 69 JF - The FEBS journal JO - FEBS J VL - 275 IS - 3 N2 - IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-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 beta-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 beta-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 beta-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. SN - 1742-464X UR - https://www.unboundmedicine.com/medline/citation/18167139/Protein_folding_intermediates_of_invasin_protein_IbeA_from_Escherichia_coli_ DB - PRIME DP - Unbound Medicine ER -