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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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Fractional contacts of β-stranded contact clusters, based on the first set of simulation data. There are four sets of multiplots under each of the columns for each flagellin subdomain, each representing a temperature. From the top of each multiplot, the change of fractional contacts with time for contact clusters are listed in the following order: β12β13, β13β14, β14β15, and β14β16 in D2a; β16β17 and β17β18 in D2b; β6β7, β7β8, β8β9, β5β7, and β6β10 in D3. Note that fractional contacts for 300 K were computed with respect to the equilibrated structure while values for 400 K and above were based on persistent native contacts obtained from the 300 K trajectory.
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fig7: Fractional contacts of β-stranded contact clusters, based on the first set of simulation data. There are four sets of multiplots under each of the columns for each flagellin subdomain, each representing a temperature. From the top of each multiplot, the change of fractional contacts with time for contact clusters are listed in the following order: β12β13, β13β14, β14β15, and β14β16 in D2a; β16β17 and β17β18 in D2b; β6β7, β7β8, β8β9, β5β7, and β6β10 in D3. Note that fractional contacts for 300 K were computed with respect to the equilibrated structure while values for 400 K and above were based on persistent native contacts obtained from the 300 K trajectory.

Mentions: Changes in fraction of persistent native contacts for D2a, D2b, and D3 under each denaturing temperature from the first set of simulations are presented in Fig. 7. For D2a at 500 K, although β14β16 is most persistent in this simulation, the top spot is taken by β13β14 among the other 500 K simulations and also in the first 600 K trajectory. The key stabilizing residue on β14 is Y332, which formed aromatic-aliphatic interactions with V325 on β13. The persistent contacts could have restricted the increase in Rg for D2a as compared to D2b at 500 K (Fig. 8). In the case of D3, though β6β7 is the most persistent at 500 K, β7β8 is also persistent at 600 K. Residues F222 and Y229 in β6β7 form favorable interaction with their side-chain aromatic rings tilted at a mean angle of 57° (from control simulation). Moreover, the lower Rg at 500 K relative to 600 K could be a result of compact region formation in the denatured state, involving hydrophobic residues 245, 247, and 229–231.


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

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

Fractional contacts of β-stranded contact clusters, based on the first set of simulation data. There are four sets of multiplots under each of the columns for each flagellin subdomain, each representing a temperature. From the top of each multiplot, the change of fractional contacts with time for contact clusters are listed in the following order: β12β13, β13β14, β14β15, and β14β16 in D2a; β16β17 and β17β18 in D2b; β6β7, β7β8, β8β9, β5β7, and β6β10 in D3. Note that fractional contacts for 300 K were computed with respect to the equilibrated structure while values for 400 K and above were based on persistent native contacts obtained from the 300 K trajectory.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Fractional contacts of β-stranded contact clusters, based on the first set of simulation data. There are four sets of multiplots under each of the columns for each flagellin subdomain, each representing a temperature. From the top of each multiplot, the change of fractional contacts with time for contact clusters are listed in the following order: β12β13, β13β14, β14β15, and β14β16 in D2a; β16β17 and β17β18 in D2b; β6β7, β7β8, β8β9, β5β7, and β6β10 in D3. Note that fractional contacts for 300 K were computed with respect to the equilibrated structure while values for 400 K and above were based on persistent native contacts obtained from the 300 K trajectory.
Mentions: Changes in fraction of persistent native contacts for D2a, D2b, and D3 under each denaturing temperature from the first set of simulations are presented in Fig. 7. For D2a at 500 K, although β14β16 is most persistent in this simulation, the top spot is taken by β13β14 among the other 500 K simulations and also in the first 600 K trajectory. The key stabilizing residue on β14 is Y332, which formed aromatic-aliphatic interactions with V325 on β13. The persistent contacts could have restricted the increase in Rg for D2a as compared to D2b at 500 K (Fig. 8). In the case of D3, though β6β7 is the most persistent at 500 K, β7β8 is also persistent at 600 K. Residues F222 and Y229 in β6β7 form favorable interaction with their side-chain aromatic rings tilted at a mean angle of 57° (from control simulation). Moreover, the lower Rg at 500 K relative to 600 K could be a result of compact region formation in the denatured state, involving hydrophobic residues 245, 247, and 229–231.

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