Folding energetics of a multidomain protein, flagellin

Thermodynamic investigations of flagellin from Salmonella typhimurium and i ts proteolytic fragments were conducted by differential scanning calorimetr y (DSC) and circular dichroism (CD) melting measurements. A new method of a nalysis for a multi-state transition based on our original theoretical trea tment of thermodynamic equations has been developed to analyze those data. The analysis of DSC curves confirmed the three thermodynamic domains of fla gellin. The thermodynamic parameters of each domain were revised from those previously reported and the new values of the parameters have a good:corre lation to the apparent molecular masses of the morphological-domains. CD me lting measurements at far and near-UV wavelengths showed sequential unfoldi ng of the domains. Therefore, we could reasonably assign the thermodynamica lly identified domains to the morphological domains. Further analysis of bo th DSC and CD data provided insights into the folding energetics of the mul tidomain structure of flagellin. An inner domain (D(f)1) of flagellin in th e filament unfolds through a relatively broad transition, while the two out er domains unfold cooperatively and show sharp transitions. This indicates that the interdomain interactions between D(f)1 and D2 has different charac teristics from the apparently more intimate interactions between D2 and D3. These characteristics suggest that flagellin is organized with relatively flexible domains and rigid domains, which appears to be responsible for the well-regulated assembly mechanism of the bacterial flagellar filament. (C) 1999 Academic Press.