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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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Radius of gyration of (A) subdomain D2a, (B) subdomain D2b, and (C) domain D3 under different temperatures from the first set of simulations: control (bold continuous), 400 K (dashed), 500 K (continuous), and 600 K (dot-dashed line with solid circles). Data points have been resampled at an interval of 20 ps.
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fig8: Radius of gyration of (A) subdomain D2a, (B) subdomain D2b, and (C) domain D3 under different temperatures from the first set of simulations: control (bold continuous), 400 K (dashed), 500 K (continuous), and 600 K (dot-dashed line with solid circles). Data points have been resampled at an interval of 20 ps.

Mentions: From the first 400 K simulation trajectory, we found that the Rg value increased gradually from 11 Å and suddenly jumped to 16 Å at ∼4 ns before dropping back at ∼12 Å (see Fig. 8 B). This spike could be due to the partial unfolding and refolding of the hydrophobic core region, with T347 separated from the A385-V373 pair. The opening-up of D2b is illustrated in Fig. S2 in the Supplementary Material, Data S1, via simulation snapshots. Combined SASAs of selected core residues from all simulations are shown in Fig. S3 in the Supplementary Material, Data S1. A threshold of 80 Å2 was adopted from the averaged SASA of polar residues reported in an early MD study (32). Fluctuations of D2b SASA at 400 K are large compared to those in the control or to those of D3 which remain folded. The above observations indicate that D2b has very low stability and would become folded only after D2a and D3.


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

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

Radius of gyration of (A) subdomain D2a, (B) subdomain D2b, and (C) domain D3 under different temperatures from the first set of simulations: control (bold continuous), 400 K (dashed), 500 K (continuous), and 600 K (dot-dashed line with solid circles). Data points have been resampled at an interval of 20 ps.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Radius of gyration of (A) subdomain D2a, (B) subdomain D2b, and (C) domain D3 under different temperatures from the first set of simulations: control (bold continuous), 400 K (dashed), 500 K (continuous), and 600 K (dot-dashed line with solid circles). Data points have been resampled at an interval of 20 ps.
Mentions: From the first 400 K simulation trajectory, we found that the Rg value increased gradually from 11 Å and suddenly jumped to 16 Å at ∼4 ns before dropping back at ∼12 Å (see Fig. 8 B). This spike could be due to the partial unfolding and refolding of the hydrophobic core region, with T347 separated from the A385-V373 pair. The opening-up of D2b is illustrated in Fig. S2 in the Supplementary Material, Data S1, via simulation snapshots. Combined SASAs of selected core residues from all simulations are shown in Fig. S3 in the Supplementary Material, Data S1. A threshold of 80 Å2 was adopted from the averaged SASA of polar residues reported in an early MD study (32). Fluctuations of D2b SASA at 400 K are large compared to those in the control or to those of D3 which remain folded. The above observations indicate that D2b has very low stability and would become folded only after D2a and D3.

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