Jacob W. Gauer

Mechanism for Calcium Ion Sensing by the C2A Domain of Synaptotagmin I

The C2A domain is one of two calcium ion (Ca(2+))- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca(2+) sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2As response to Ca(2+) and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca(2+)- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca(2+)- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.

Negative Coupling as a Mechanism for Signal Propagation between C2 Domains of Synaptotagmin I

Synaptotagmin I (Syt I) is a vesicle-localized protein implicated in sensing the calcium influx that triggers fast synchronous release of neurotransmitter. How Syt I utilizes its two C2 domains to integrate signals and mediate neurotransmission has continued to be a controversial area of research, though prevalent hypotheses favor independent function. Using differential scanning calorimetry and fluorescence lifetime spectroscopy in a thermodynamic denaturation approach, we tested an alternative hypothesis in which both domains interact to cooperatively disseminate binding information. The free energy of stability was determined for C2A, C2B, and C2AB constructs by globally fitting both methods to a two-state model of unfolding. By comparing the additive free energies of C2A and C2B with C2AB, we identified a negative coupling interaction between the C2 domains of Syt I. This interaction not only provides a mechanistic means for propagating signals, but also a possible means for coordinating the molecular events of neurotransmission.

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²⁺) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²⁺ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²⁺ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²⁺ affinity to a membrane-associated, higher Ca²⁺ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²⁺ influx is the basis for the cooperative response of annexin a5 toward Ca²⁺, and the role of membrane organization in this response.

Original research in the classroom: Why do zebrafish spawn in the morning?

As part of an upper level undergraduate developmental biology course at the University of Minnesota Duluth, we developed a unit in which students carried out original research as part of a cooperative class project. Students had the opportunity to gain experience in the scientific method from experimental design all of the way through to the preparation of publication on their research that included text, figures, and tables. This kind of inquiry-based learning has been shown to have many benefits for students, including increased long-term learning and a better understanding of the process of scientific discovery. In our project, students designed experiments to explore why zebrafish typically spawn in the first few hours after the lights come on in the morning. The results of our experiments suggest that spawning still occurs when the dark-to-light transition is altered or absent. This is consistent with the work of others that demonstrates that rhythmic spawning behavior is regulated by an endogenous circadian clock. Our successes and failures carrying out original research as part of an undergraduate course should contribute to the growing approaches for using zebrafish to bring the excitement of experimental science to the classroom.

Ligand Induced Conformational Redistribution in Synaptotagmin I C2A

Thermodynamic parameters capture the overall contribution to a systems energetics. In the case of binding proteins, such as Synaptotagmin I, ascertaining the overall magnitude of the interactions within the protein is the first step toward addressing how energy is distributed throughout the body of the protein. Our aim is to understand how the signal of ligand binding is disseminated through the protein during the role it plays in regulated exocytosis. While several detailed molecular approaches have identified putative regions where interactions occur, it is the energetics that are key to understanding Synaptotagmin Is functional response. Here, denaturation studies of the C2A domain of Synaptotagmin I were carried out in conditions that are physiologically relevant to regulated exocytosis where calcium ions and phospholipids were either present or absent. Denaturation was carried out using two techniques: differential scanning calorimetry (DSC) and fluorescence lifetime (FLT). A global analysis approach combining these data sets was used where the data was simultaneously fit to models derived from thermodynamic principles. The enthalpy associated with the denaturation of the C2A domain of Synaptotagmin I in the absence of all ligands was found to be quite low when compared to other proteins of the similar molecular weight, which suggests that the protein exhibits conformational flexibility. In addition, the denaturation behavior is shown to change when ligand is bound, which suggest a similar change in the conformational flexibility.

Utilization of Thermodynamic Linkage Relationships to Test for Interactions between the C2 Domains of Synaptotagmin 1

Synaptotagmin I is a calcium ion sensor involved in neurotransmitter release. When a neuron is stimulated by an action potential, an influx of calcium ion occurs causing the synaptic vesicle to fuse with the membrane, releasing the neurotransmitter into the synaptic cleft. This causes a new action potential to propagate to the next neuron, thereby transmitting information from cell to cell by electrochemical signaling. Synaptotagmin I is characterized by a transmembrane region embedded in the synaptic vesicle at its N-terminus which is connected to two C2 domains (C2A and C2B) that are located in the cytoplasm of the neuron. Both C2 domains bind calcium ions, negatively charged phospholipids, and associate with other proteins that are also involved with neurotransmitter release. Whether or not this process is mediated by a protein-protein interaction between the C2 domains is unknown. Since these proteins are tethered together, the very high effective concentration of one in relation to the other means the interaction energies must be very small; otherwise, the proteins would always be associated. Transient interactions represent a means to communicate binding information. Testing this is extremely challenging due to weak interactions. Here, we have utilized a thermal denaturation approach dependent on determination of free energies of stability to test this hypothesis.