Aquifex aeolicus FlgM protein exhibits a temperature-dependent disordered nature

Abstract

Studies on the nature and function of intrinsically disordered proteins (IDP) over the past 10 years have demonstrated the importance of IDPs in normal cellular function. Although many proteins predicted to be IDPs have been experimentally characterized on an individual basis, the conservation of disorder between homologous proteins from different organisms has not been fully studied. We now demonstrate that the FlgM protein from the thermophile Aquifex aeolicus exhibits a more ordered conformation at 20 degrees C than the previously characterized FlgM protein from Salmonella typhimurium. FlgM is an inhibitor of the RNA transcription factor sigma28, which is involved in regulation of the late-stage genes involved in flagella synthesis. Previous work has shown that the S. typhimurium FlgM protein is an intrinsically disordered protein, though the C-terminus becomes ordered when bound to sigma28 or under crowded solution conditions. In this work, we demonstrate that at 20 degrees C the A. aeolicus FlgM protein exhibits alpha-helical character in circular dichroism (CD) experiments, though the percentage of alpha-helical content decreases with increased temperature, consistent with the FlgM assuming a less folded conformation. We also show that the A. aeolicus FlgM exhibits cooperativity in chemical denaturation experiments, consistent with a globular nature. Furthermore, we use the fluorescent probe FlAsH to show that the H2 helix is ordered, even in the unbound state and that the H1 and H2 helices appear to be associated with each other in the absence of the sigma28 protein. Finally, we demonstrate that the H2 helix assumes an extended conformation at 85 degrees C. Based on our results, we propose that at 20 degrees C the A. aeolicus FlgM assumes a four-helix bundle-like conformation that becomes a more extended conformation at the A. aeolicus' physiological temperature of 85 degrees C.