Thermal perturbation difference spectroscopy of [formula omitted] flagellin

Abstract

The appropriateness of a two-state model for the previously reported thermal transition of Salmonella flagellin and the reversibility of this process recently has been questioned by others. Spectrophotometric evidence is presented here that reveals two separate thermally-induced structural transitions in flagellin which may resolve apparent controversy. A low temperature transition (I) appears between 28 and 35°C with a midtransition temperature of 30.7°C. With increasing temperature in this transition region a progressive shift to the red of the absorbance band at 284 nm occurs. The latter, probably due to an infolding of tyrosyl residues, is paralleled by a decrease in the rate of polymerization of flagellin. The temperature profile for the spectral behavior of flagellin in transition I fulfills criteria for a two-state process and is fully reversible. A second transition, also reversible, is observed between 40 and 60°C with a midtransition temperature near 50°C. Transition II, observed as a blue spectral shift of the absorbance band at 277 nm, is better described as a multistate process. Tyrosyl residues appear to maintain the conformational integrity of the polymerizable state of flagellin.