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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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Related in: MedlinePlus

Representative trajectory snapshots taken from the first 500 K thermal unfolding simulation (after root-mean-square fitting on D3 residues), presented in reversed time order to show folding of domain D3. The N- and C-termini are labeled. Folding starts from stabilization of the arch-loop conformation by proline-aromatic interactions between pairs P265-Y245 (magenta sticks) and P271-Y230 (blue sticks) across the loop and the β-folium sheet, denoting event d in Fig. 5. Aromatic-aromatic interaction between F222 and Y190 (on terminal β-sheet, which is also the D2-D3 linker) shown here as orange sticks helps to bind the terminal sheet against the rest of the domain (event c). The tight packing of F202 in the hydrophobic core (F202, V233, and V247 shown as light green sticks) takes the longest time (event b). Note the formation of the terminal β-sheet after F222-Y190 interaction becomes stable. Molecular scenes rendered by Tachyon ray tracer (53) within VMD.
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fig6: Representative trajectory snapshots taken from the first 500 K thermal unfolding simulation (after root-mean-square fitting on D3 residues), presented in reversed time order to show folding of domain D3. The N- and C-termini are labeled. Folding starts from stabilization of the arch-loop conformation by proline-aromatic interactions between pairs P265-Y245 (magenta sticks) and P271-Y230 (blue sticks) across the loop and the β-folium sheet, denoting event d in Fig. 5. Aromatic-aromatic interaction between F222 and Y190 (on terminal β-sheet, which is also the D2-D3 linker) shown here as orange sticks helps to bind the terminal sheet against the rest of the domain (event c). The tight packing of F202 in the hydrophobic core (F202, V233, and V247 shown as light green sticks) takes the longest time (event b). Note the formation of the terminal β-sheet after F222-Y190 interaction becomes stable. Molecular scenes rendered by Tachyon ray tracer (53) within VMD.

Mentions: From the thermal unfolding simulations, we identified key events during the unfolding of domain D3. In the state-diagram (Fig. 5), we present the order of these events (local events b–d) as observed from each simulation. Conformation snapshots of D3 unfolding are presented in reversed time order in Fig. 6.


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

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

Representative trajectory snapshots taken from the first 500 K thermal unfolding simulation (after root-mean-square fitting on D3 residues), presented in reversed time order to show folding of domain D3. The N- and C-termini are labeled. Folding starts from stabilization of the arch-loop conformation by proline-aromatic interactions between pairs P265-Y245 (magenta sticks) and P271-Y230 (blue sticks) across the loop and the β-folium sheet, denoting event d in Fig. 5. Aromatic-aromatic interaction between F222 and Y190 (on terminal β-sheet, which is also the D2-D3 linker) shown here as orange sticks helps to bind the terminal sheet against the rest of the domain (event c). The tight packing of F202 in the hydrophobic core (F202, V233, and V247 shown as light green sticks) takes the longest time (event b). Note the formation of the terminal β-sheet after F222-Y190 interaction becomes stable. Molecular scenes rendered by Tachyon ray tracer (53) within VMD.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Representative trajectory snapshots taken from the first 500 K thermal unfolding simulation (after root-mean-square fitting on D3 residues), presented in reversed time order to show folding of domain D3. The N- and C-termini are labeled. Folding starts from stabilization of the arch-loop conformation by proline-aromatic interactions between pairs P265-Y245 (magenta sticks) and P271-Y230 (blue sticks) across the loop and the β-folium sheet, denoting event d in Fig. 5. Aromatic-aromatic interaction between F222 and Y190 (on terminal β-sheet, which is also the D2-D3 linker) shown here as orange sticks helps to bind the terminal sheet against the rest of the domain (event c). The tight packing of F202 in the hydrophobic core (F202, V233, and V247 shown as light green sticks) takes the longest time (event b). Note the formation of the terminal β-sheet after F222-Y190 interaction becomes stable. Molecular scenes rendered by Tachyon ray tracer (53) within VMD.
Mentions: From the thermal unfolding simulations, we identified key events during the unfolding of domain D3. In the state-diagram (Fig. 5), we present the order of these events (local events b–d) as observed from each simulation. Conformation snapshots of D3 unfolding are presented in reversed time order in Fig. 6.

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
Related in: MedlinePlus