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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) Changes to persistent contact maps from the first set of simulations. Here, only persistent contacts within each time window of 2-ns (400 K), 0.5-ns (500 K), or 0.2-ns (600 K) are plotted. (B) Changes to DSSP assigned secondary structures from the same simulation set (black, β-strand; gray, α-helix), with plot for 300 K shown as inset to Fig. 2. Note that simulation lengths differ: 8-ns (400 K), 6-ns (500 K), and 2-ns (600 K).
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fig3: (A) Changes to persistent contact maps from the first set of simulations. Here, only persistent contacts within each time window of 2-ns (400 K), 0.5-ns (500 K), or 0.2-ns (600 K) are plotted. (B) Changes to DSSP assigned secondary structures from the same simulation set (black, β-strand; gray, α-helix), with plot for 300 K shown as inset to Fig. 2. Note that simulation lengths differ: 8-ns (400 K), 6-ns (500 K), and 2-ns (600 K).

Mentions: Time-averaged persistent contact maps from the first set of simulations indicated that pairs of β-strands in domains D2 and D3 remained for varying simulation lengths depending on the temperature (Fig. 3 A). Corresponding secondary structure changes in Fig. 3 B also revealed persistent β-strands in D2 and D3 while α-helices in D0 and D1 became denatured. To facilitate subsequent discussions, we need to introduce domain fragment Df1. This is the proteolysis-resistant portion of D1, which includes not just residues from the N-terminal side as originally defined (8) but also from the C-terminal side, as marked on the contact map (Fig. 2 A). Df1, colored black in Fig. 2 B, contains an elongated hydrophobic core that could account for its proteolytic resistance. The rigidity of Df1 hydrophobic core has been noted in a simulation of a 44-mer model of the filament (31). The remaining fragment of D1, colored magenta, is indeed found to be less structured during our simulations (Fig. 4).


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

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

(A) Changes to persistent contact maps from the first set of simulations. Here, only persistent contacts within each time window of 2-ns (400 K), 0.5-ns (500 K), or 0.2-ns (600 K) are plotted. (B) Changes to DSSP assigned secondary structures from the same simulation set (black, β-strand; gray, α-helix), with plot for 300 K shown as inset to Fig. 2. Note that simulation lengths differ: 8-ns (400 K), 6-ns (500 K), and 2-ns (600 K).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: (A) Changes to persistent contact maps from the first set of simulations. Here, only persistent contacts within each time window of 2-ns (400 K), 0.5-ns (500 K), or 0.2-ns (600 K) are plotted. (B) Changes to DSSP assigned secondary structures from the same simulation set (black, β-strand; gray, α-helix), with plot for 300 K shown as inset to Fig. 2. Note that simulation lengths differ: 8-ns (400 K), 6-ns (500 K), and 2-ns (600 K).
Mentions: Time-averaged persistent contact maps from the first set of simulations indicated that pairs of β-strands in domains D2 and D3 remained for varying simulation lengths depending on the temperature (Fig. 3 A). Corresponding secondary structure changes in Fig. 3 B also revealed persistent β-strands in D2 and D3 while α-helices in D0 and D1 became denatured. To facilitate subsequent discussions, we need to introduce domain fragment Df1. This is the proteolysis-resistant portion of D1, which includes not just residues from the N-terminal side as originally defined (8) but also from the C-terminal side, as marked on the contact map (Fig. 2 A). Df1, colored black in Fig. 2 B, contains an elongated hydrophobic core that could account for its proteolytic resistance. The rigidity of Df1 hydrophobic core has been noted in a simulation of a 44-mer model of the filament (31). The remaining fragment of D1, colored magenta, is indeed found to be less structured during our simulations (Fig. 4).

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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