For the delivery of signaling molecules in a cell to occur, the membranes of the vesicle in which signaling molecules are packaged must fuse with another membrane. As timing is vital in cellular processes, this delivery of signaling molecules must be poised to trigger only when the appropriate signal or combinations of cellular signals transpire. Although this is a critical cellular event, how this regulated delivery occurs is not well understood. Membranes are comprised of a great diversity of lipids whose individual lipid-lipid interactions are weak and interactions within the membrane dictated by the lipids' chemical structure and its local milieu. These factors govern the resultant lipid's activity (effective concentration) and hence, how the membrane is targeted for binding to or partitioning into by proteins. Within this model, it is hypothesized that intrinsically weak interactions between lipids are critical for the system to reversibly and rapidly organize the protein clusters that modulate signaling. This offers a more nuanced regulation of biological events beyond a system dominated by a few, very strong interactions. These small interaction energies offer flexibility in how combinations of lipids, in response to proteins binding, form signaling platforms. This concept is hypothesized to be the basis for how key proteins involved in regulated delivery target distinct membranes. The objective of this project is to test the hypothesis that these key proteins are modulated not by a few strong interactions but by a necessary ensemble of weak interactions at the membrane. Testing will involve quantitative analysis and rigorous use of binding theory and thermodynamic (linkage) relationships by undergraduates from diverse scientific disciplines. New techniques, both conceptual and experimental, will also result from this project that will be applicable to many questions of biological relevance. The classic use of linkage relationships is an example of problem-based learning where biological processes are simply represented mathematically and interpreted. Each experiment becomes a case study and the derivation of equations is no longer restricted to assigned homework or to be used after years of graduate training. The goal is to demonstrate that undergraduates and young graduate students can propose, use, explore, and test these concepts.
Broader Impacts The Broader Impacts of this project include the development of methodologies that will enhance the persistence, retention, and future success of women in scientific careers. By improving the educational environment for women, the educational environment not only for underrepresented groups but for all students will be improved. The methodologies will have two components, Informal and Formal Education. The Informal structure for this change is a scientific council, named Interactions. This council will integrate all issues that are faced by future science, mathematics and engineering (SME) professionals. Potential topics will include: social dynamics (e.g. bias), the importance of building a community in addition to a resume (or CV), the need to apply for research scholarships, academic scholarships and awards, and how these experiences can shape your future. Interactions will provide in-house educational meetings and host motivating role models such as distinguished female scientists, alumni, and scientists from the region who have gone on to illustrious careers. Interactions will also foster peer group learning. The existence of peer group learning has been identified by SME students as having the most immediate and most effective contribution to increasing persistence of students within SME majors. The Formal component is a Physical Biochemistry capstone course with laboratory capturing the convergence of topics across the major and explicitly uniting concepts through application. The linkage of biochemistry with other disciplines will be through quantitative literacy where the grammar is Physical Chemistry. A course focused upon practical outcomes will emphasize female specific advantages in learning as it is argued that many other learning strategies are inherently favorable to male learning patterns. Gender differences in learning strategies will be equalized through multiple approaches to problem solving, and the commonalities between seemingly diverse topics will be emphasized.
The synaptic vesicle protein Synaptotagmin I (Syt I) is the calcium ion sensor for neurotransmitter release. The very act of thought and response, of decision and action converge on this protein sensing an influx of calcium ion by interacting with (binding to) this potent signaling molecule and conveying this message to act. While Syt I has been subject to intense scrutiny, how it translates and propagates the calcium ion signal with the plasticity seemingly necessary for literally a thoughtful response is not clear. We propose that Syt I acts as a unified protein rather than as a combination of independent binding modules (domains). A Syt I protein is comprised of two C2 domains, calcium ion dependent, membrane-binding domains, which are tethered in series to the membrane. The basis for our proposed model resides upon our finding that each of the binding domains of Syt I are nearly disordered. While its crystallographic structure may appear to have well-defined structure, experimentally defined thermodynamic parameters suggest otherwise. They suggest a protein that is malleable, a potential mechanism of response. The energetic measures of the interactions that glue the combined or separate C2 domains together were low, shockingly low. Moreover, upon binding of calcium ion, each C2 domain became more stable, consistent with a more defined set of interactions compared to when not bound. When the C2 domains were united, they were found to have even less energetic interactions holding them into a neatly folded state. When attached to one another, the C2 domains melted (destabilized) each other so that each C2 domain could more easily access different forms (conformations) of each C2 domain. If different conformations correlate with different functional outcomes, plasticity in protein conformations will correlate with plasticity in protein response. This interpretation of the thermodynamic data appears in keeping with a protein that is primary to thought and feeling. This response is nuanced. When each C2 domain binds a lipid to which it exhibits specificity, the binding C2 domain becomes more stable and the adjacent C2 domains becomes less stable. This is a means to communicate information; it is a form of crosstalk. The unstable C2 domain is a more responsive protein platform as it can sample more conformations and hence, encompasses the possibility of more functional outcomes than the more stable form that exists prior to neighboring domain receiving the information transmitted by binding. A protein with such plasticity also offers the potential for a cooperative response. If the protein can sample all the conformations with a relatively low energetic barrier between each upon binding of calcium ion, the unbound conformation that had specificity for calcium ion is depleted. The equilibrium shifts to replenish that depleted form, essentially driving the conformations into existing primarily in the calcium ion bound state. The resultant depletion of the other conformers when combined with binding of multiple calcium ions is the basis of this cooperative response. Our proposed model for the basis of how Synaptotagmin I responds to calcium ion to culminate in neurotransmitter release extends well beyond human thought and emotion. Calcium ion influx is a common means to communicate information in eukaryotes and there are families of eukaryotic proteins that are calcium-stimulated membrane binding proteins. Is malleability as defined by marginal stability (near disorder), a common means for these types of proteins to integrate these two seemingly disparate signals? Thus far, the hallmark of marginal stability was found in all C2 domains tested whether from plant or from animal. Marginal stability was not the only common thermodynamic signature defined. Like the synaptotagmins, the annexins, also ancient denizens of eukaryotic life and a many membered family, respond to both calcium ion and membrane but are structurally distinct. We propose a common mechanism as to how these different protein families integrate the lipid distributions of each local membrane surface with calcium ion influx. Rather than being a calcium-stimulated, membrane binding protein, we propose that both the synaptotagmins and annexins are weakly membrane-associated prior to a calcium ion influx as the basis of a membrane-defined, cooperative response. The membrane-induced conformation of these proteins has greater calcium ion affinity than the much more predominant solution form of the protein. Thus, upon influx of calcium ion, some of the membrane-associated form of these proteins binds calcium ion due to their enhanced affinity. This form is repopulated, drawing the equilibrium forward to deplete all other conformers in favor of the membrane-associated, calcium ion binding conformation. The consequent lag and then abrupt rise in the population of protein that is fully bound to calcium ion is the hallmark of a biologic switch, of cooperative binding. A certain degree of malleability and of plasticity underlies such a membrane-defined response to calcium ion, plasticity common to the membrane itself and the distribution of lipids within it.
