Conformational flexibility of escherichia coli Hsp31 protein

Abstract

The mRNA of Escherichia coli yedU gene is induced upon heat shock. The 31kDa YedU protein is a homodimeric protein consisting of a large A domain and a smaller P domain connected by a linker. Especially, the P domain is responsible for dimer formation through inter-subunit interactions. Crystal structures of YedU indicate that it exists in two different forms. The flexible region, D2 and D3, of form I are ordered, whereas that of form II is disordered. The flexibility in a number of intra-domain linker regions found in the form II indicates a role of YedU in chaperone action. With the purified protein, we detected two major peaks of YedU protein, separated by anion-exchange chromatography, while catalytic mutants, E77A, C185A, C185D, and H186A, generated only the first peak, which is consistent with iso-electric focusing result. To gain further insight into their conformation, we measured the circular dichroism (CD) spectra of different YedU peaks, in which peak 1 contains α-helical and ß-sheet structures of 26% and 24%, respectively, and peak 2 with 16% α-helix and 32% ß-sheet. Based on three tryptophan residues of YedU, the equilibrium folding behaviors of YedU protein were examined by fluorescence spectroscopy. The peak 1 of YedU exhibited a fluorescence maximum at 348 nm, whereas the peak 2 exhibited a much lower intensity with maximum at 336 nm. Although we did not detect remarkable drop of fluorescence intensity, maximum fluorescence $(λ_{max})$ shifts to shorter wavelength as GdnHCl concentration or temperature increases. The equilibrium unfolding data indicates two-state transition involving the native and unfolded states. Although the CD data exhibits slightly different secondary structural contents from the calculated one of crystal structure, it is likely that the peak 1 and peak 2 represent two different conformational states of YedU due to a flexibility involving the D2 region.