Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Thursday Speaker Abstracts

Synaptic Vesicle Lipids Reveal Structured State and Antagonistic Allosteric Mechanisms of Intrinsic Disorder within Synaptotagmin I Michael E. Fealey 1,2 , Anne Hinderliter 2 . 1 University of Minnesota, Minneapolis, MN, USA, 2 University of Minnesota Duluth, Duluth, MN, USA. Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein responsible for sensing the calcium influx that triggers neurotransmitter release. Syt I consists of a transmembrane helix, a cytosolic 60 residue tether region, and two C2 domains that bind calcium and acidic phospholipids. Until recently, the role of the 60 residue tether region in Syt I function received little attention. We noticed that this tether region has features of intrinsic disorder and hypothesized that it exerts allosteric control over the adjacent calcium binding C2 domains to tune protein function. In testing this hypothesis, we first assessed the impact of local lipid environment on the structure of the isolated tether region. Using differential scanning calorimetry (DSC) and nuclear magnetic resonance, we found that a lipid composition mimicking a synaptic vesicle selects for ordered conformers in the intrinsically disordered sequence. A simple binary lipid mixture did not have any apparent ordering impact. Knowing the intrinsically disorder region was influenced by a membrane that mimics its native organelle, we next assessed its allosteric impact on the first C2 domain (C2A) using DSC. Strikingly, we found that discrete regions of the disordered tether had opposite effects on C2A unfolding. Calcium binding to C2A in the presence of the entire disordered region shifts the unfolding transition to low temperature. In contrast, when the 13 most N-terminal residues (mostly lysines) are removed, calcium binding dramatically increases the temperature of the unfolding transition. In both cases, calcium binding stabilizes the protein but the extent differs as do the contributions of each thermodynamic parameter underlying the transition. These results indicate that the combine intrinsic disorder and synaptic vesicle lipids have the ability to evoke antagonistic responses of Syt I to calcium ligation.

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