Thermal Unfolding Simulations of Bacterial Flagellin: Insight into its Refolding before Assembly

¹ University of Tokyo

^* To whom correspondence should be addressed. E-mail: kitao{at}iam.u-tokyo.ac.jp.

Submitted on October 15, 2007
Revised on November 17, 2007
Accepted on 10 January 2008

	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 (HVR) 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 -sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagensis 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 HVR domains or filament-core domains but not whole flagellin, implicating a two-staged refolding process.

Key Words: Type-III secretion system, denatured state, folding core, molecular dynamics, multi-domain, protein translocation