Limits...
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.

Show MeSH

Related in: MedlinePlus

A symbolic representation of major events in the thermal unfolding of flagellin from all simulations conducted in this study. The criteria used were as follows. States 1–4: when the 200-ps running average of the hybrid metric (see text for definition) first dropped below the 0.2 threshold. Fractional secondary structure content at time t in a trajectory is defined as the number of residues assigned as helix (Df1) or strand (D2a, D2b, D3) divided by the maximum number from the whole trajectory. State a: when the maximum instantaneous residue SASA among the D2b hydrophobic core residues first exceeded 30 Å2, which is the maximum observed during the control simulation. State b: similar to state a, but only one residue is considered and threshold is 25 Å2. State c: when minimum heavy atom separation of Y190 and F222 first exceeded the contact threshold without subsequent fluctuations about the threshold. States d and e: when the 200-ps running-averaged fractional native contact dropped below 0.2.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2367190&req=5

fig5: A symbolic representation of major events in the thermal unfolding of flagellin from all simulations conducted in this study. The criteria used were as follows. States 1–4: when the 200-ps running average of the hybrid metric (see text for definition) first dropped below the 0.2 threshold. Fractional secondary structure content at time t in a trajectory is defined as the number of residues assigned as helix (Df1) or strand (D2a, D2b, D3) divided by the maximum number from the whole trajectory. State a: when the maximum instantaneous residue SASA among the D2b hydrophobic core residues first exceeded 30 Å2, which is the maximum observed during the control simulation. State b: similar to state a, but only one residue is considered and threshold is 25 Å2. State c: when minimum heavy atom separation of Y190 and F222 first exceeded the contact threshold without subsequent fluctuations about the threshold. States d and e: when the 200-ps running-averaged fractional native contact dropped below 0.2.

Mentions: The sole use of fractional contacts to determine whether a (sub)domain is denatured does not work well for D2b, as the value can remain >0.1 while loss of secondary structures continues (data not shown). The situation is reversed for Df1. To capture both aspects of denaturation, we introduce a hybrid metric: the square-root of the product of fractional native contacts and fractional secondary structure content. When the 200-ps running average of this metric first dropped to <0.2, a value somewhat arbitrarily assigned, we say that the (sub)domain has denatured. Using these criteria to assign the global unfolding events and other criteria for the local unfolding events, we constructed a state-diagram for all the simulations as shown in Fig. 5. We noticed a general conservation of global unfolding events, such as D2b and D2a being the first and last to denature, respectively.


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

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

A symbolic representation of major events in the thermal unfolding of flagellin from all simulations conducted in this study. The criteria used were as follows. States 1–4: when the 200-ps running average of the hybrid metric (see text for definition) first dropped below the 0.2 threshold. Fractional secondary structure content at time t in a trajectory is defined as the number of residues assigned as helix (Df1) or strand (D2a, D2b, D3) divided by the maximum number from the whole trajectory. State a: when the maximum instantaneous residue SASA among the D2b hydrophobic core residues first exceeded 30 Å2, which is the maximum observed during the control simulation. State b: similar to state a, but only one residue is considered and threshold is 25 Å2. State c: when minimum heavy atom separation of Y190 and F222 first exceeded the contact threshold without subsequent fluctuations about the threshold. States d and e: when the 200-ps running-averaged fractional native contact dropped below 0.2.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: A symbolic representation of major events in the thermal unfolding of flagellin from all simulations conducted in this study. The criteria used were as follows. States 1–4: when the 200-ps running average of the hybrid metric (see text for definition) first dropped below the 0.2 threshold. Fractional secondary structure content at time t in a trajectory is defined as the number of residues assigned as helix (Df1) or strand (D2a, D2b, D3) divided by the maximum number from the whole trajectory. State a: when the maximum instantaneous residue SASA among the D2b hydrophobic core residues first exceeded 30 Å2, which is the maximum observed during the control simulation. State b: similar to state a, but only one residue is considered and threshold is 25 Å2. State c: when minimum heavy atom separation of Y190 and F222 first exceeded the contact threshold without subsequent fluctuations about the threshold. States d and e: when the 200-ps running-averaged fractional native contact dropped below 0.2.
Mentions: The sole use of fractional contacts to determine whether a (sub)domain is denatured does not work well for D2b, as the value can remain >0.1 while loss of secondary structures continues (data not shown). The situation is reversed for Df1. To capture both aspects of denaturation, we introduce a hybrid metric: the square-root of the product of fractional native contacts and fractional secondary structure content. When the 200-ps running average of this metric first dropped to <0.2, a value somewhat arbitrarily assigned, we say that the (sub)domain has denatured. Using these criteria to assign the global unfolding events and other criteria for the local unfolding events, we constructed a state-diagram for all the simulations as shown in Fig. 5. We noticed a general conservation of global unfolding events, such as D2b and D2a being the first and last to denature, 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
Related in: MedlinePlus