Calcineurin in a Crowded World

[Image: see text] Calcineurin is a Ser/Thr phosphatase that is important for key biological processes, including immune system activation. We previously identified a region in the intrinsically disordered regulatory domain of calcineurin that forms a critical amphipathic α-helix (the “distal helix”) that is required for complete activation of calcineurin. This distal helix was shown to have a T(m) close to that of human body temperature. Because the T(m) was determined in dilute buffer, we hypothesized that other factors inherent to a cellular environment might modulate the stability of the distal helix. One such factor that contributes to stability in other proteins is macromolecular crowding. The cell cytoplasm is comprised of up to 400 g/L protein, lipids, nucleic acids, and other compounds. We hypothesize that the presence of such crowders could increase the thermal stability of the distal helix and thus lead to a more robust activation of calcineurin in vivo. Using biophysical and biochemical approaches, we show that the distal helix of calcineurin is indeed stabilized when crowded by the synthetic polymers dextran 70 and ficoll 70, and that this stabilization of the distal helix increases the activity of calcineurin.