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Thermal unfolding simulations of bacterial flagellin: insight into its refolding before assembly.

Chng CP, Kitao A - Biophys. J. (2008)

Bottom Line: We observed a similar unfolding order of the domains as reported in experimental thermal denaturation.A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores.Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.

View Article: PubMed Central - PubMed

Affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded beta-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.

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(A) Residue contact map of native flagellin structure. Shaded circles indicate contacts in 1UCU. Black diamonds indicate persistent native contacts: contacts found in >70% of the snapshots taken from the last 1 ns of the control simulation as a monomer in solution. Dotted lines mark location of Df1 contact clusters (see Text). (B) The tertiary structure of flagellin monomer in solution obtained from MD simulation (starting from polymeric flagellin structure) showing DSSP-assigned α-helices and β-strands. Each (sub)domain is colored differently and labeled, with linkers colored gray. N-terminus (blue) and C-terminus (red) helices are disordered in the monomer. We have neglected the very small β-turn from residues 130 to 135 in our labeling scheme. Segment Df1 of D1 is in black, with the remaining portion in magenta. (C) The residues in the HVR domains (D2 and D3) forming persistent pairs of residue contacts in at least two of the five 500 K simulations are shown as sticks and colored by chemical element: carbon (green), oxygen (red), and nitrogen (blue). The β-sheets β6β7β8 and β12β13β14 might form folding cores. Molecular structures are rendered with PyMOL (52).
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fig2: (A) Residue contact map of native flagellin structure. Shaded circles indicate contacts in 1UCU. Black diamonds indicate persistent native contacts: contacts found in >70% of the snapshots taken from the last 1 ns of the control simulation as a monomer in solution. Dotted lines mark location of Df1 contact clusters (see Text). (B) The tertiary structure of flagellin monomer in solution obtained from MD simulation (starting from polymeric flagellin structure) showing DSSP-assigned α-helices and β-strands. Each (sub)domain is colored differently and labeled, with linkers colored gray. N-terminus (blue) and C-terminus (red) helices are disordered in the monomer. We have neglected the very small β-turn from residues 130 to 135 in our labeling scheme. Segment Df1 of D1 is in black, with the remaining portion in magenta. (C) The residues in the HVR domains (D2 and D3) forming persistent pairs of residue contacts in at least two of the five 500 K simulations are shown as sticks and colored by chemical element: carbon (green), oxygen (red), and nitrogen (blue). The β-sheets β6β7β8 and β12β13β14 might form folding cores. Molecular structures are rendered with PyMOL (52).

Mentions: In Fig. 2 B, we show a model of the four-domain, 494-residue monomeric S. typhimurium flagellin with unstructured helices in domain D0, obtained from the polymeric form (1UCU) by running a short 1.4-ns equilibration MD simulation in solvent. Domains D0 and D1 are involved with formation of the inner and outer filament tubes respectively (filament-core domains). Domains D2 (composed of subdomains D2a and D2b) and D3 make up the HVR that is exposed on the filament exterior (3). The N-terminal part of D1 (ND1) contains α2, α3, β1, and β2. The C-terminal part (CD1) consists of α6 only. Similarly, ND0 and CD0 are made up of α1 and α7, respectively.


Thermal unfolding simulations of bacterial flagellin: insight into its refolding before assembly.

Chng CP, Kitao A - Biophys. J. (2008)

(A) Residue contact map of native flagellin structure. Shaded circles indicate contacts in 1UCU. Black diamonds indicate persistent native contacts: contacts found in >70% of the snapshots taken from the last 1 ns of the control simulation as a monomer in solution. Dotted lines mark location of Df1 contact clusters (see Text). (B) The tertiary structure of flagellin monomer in solution obtained from MD simulation (starting from polymeric flagellin structure) showing DSSP-assigned α-helices and β-strands. Each (sub)domain is colored differently and labeled, with linkers colored gray. N-terminus (blue) and C-terminus (red) helices are disordered in the monomer. We have neglected the very small β-turn from residues 130 to 135 in our labeling scheme. Segment Df1 of D1 is in black, with the remaining portion in magenta. (C) The residues in the HVR domains (D2 and D3) forming persistent pairs of residue contacts in at least two of the five 500 K simulations are shown as sticks and colored by chemical element: carbon (green), oxygen (red), and nitrogen (blue). The β-sheets β6β7β8 and β12β13β14 might form folding cores. Molecular structures are rendered with PyMOL (52).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2367190&req=5

fig2: (A) Residue contact map of native flagellin structure. Shaded circles indicate contacts in 1UCU. Black diamonds indicate persistent native contacts: contacts found in >70% of the snapshots taken from the last 1 ns of the control simulation as a monomer in solution. Dotted lines mark location of Df1 contact clusters (see Text). (B) The tertiary structure of flagellin monomer in solution obtained from MD simulation (starting from polymeric flagellin structure) showing DSSP-assigned α-helices and β-strands. Each (sub)domain is colored differently and labeled, with linkers colored gray. N-terminus (blue) and C-terminus (red) helices are disordered in the monomer. We have neglected the very small β-turn from residues 130 to 135 in our labeling scheme. Segment Df1 of D1 is in black, with the remaining portion in magenta. (C) The residues in the HVR domains (D2 and D3) forming persistent pairs of residue contacts in at least two of the five 500 K simulations are shown as sticks and colored by chemical element: carbon (green), oxygen (red), and nitrogen (blue). The β-sheets β6β7β8 and β12β13β14 might form folding cores. Molecular structures are rendered with PyMOL (52).
Mentions: In Fig. 2 B, we show a model of the four-domain, 494-residue monomeric S. typhimurium flagellin with unstructured helices in domain D0, obtained from the polymeric form (1UCU) by running a short 1.4-ns equilibration MD simulation in solvent. Domains D0 and D1 are involved with formation of the inner and outer filament tubes respectively (filament-core domains). Domains D2 (composed of subdomains D2a and D2b) and D3 make up the HVR that is exposed on the filament exterior (3). The N-terminal part of D1 (ND1) contains α2, α3, β1, and β2. The C-terminal part (CD1) consists of α6 only. Similarly, ND0 and CD0 are made up of α1 and α7, respectively.

Bottom Line: We observed a similar unfolding order of the domains as reported in experimental thermal denaturation.A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores.Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.

View Article: PubMed Central - PubMed

Affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded beta-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.

Show MeSH