We propose purchasing a light scattering instrument to characterize the oligomeric states of S100 proteins. The long-term goal of the funded parent project is to reveal the binding partners for the proteins S100A5 and S100A6. These proteins bind to downstream target proteins in the cell in response to calcium; however, the precise targets remain poorly described. S100A5 and S100A6 are both upregulated in disease states, including various cancers and heart disease; however, we do not understand the causes or consequences of this effect because we do not know their binding partners. The parent project can be envisioned in two phases.
The first (Aims 1 and 2) is to identify new targets for S100A5 and S100A6 using a combination of high-throughput characterization and evolutionary analysis. The second phase (Aim 3) involves understanding the biochemical mechanisms by which S100A5 and S100A6 recognize their binding targets. We have identified putative new targets for both proteins and have started work on Aim 3. We have found that S100A5 and S100A6 radically change their oligomeric state in response to changes in solution conditions, from dimers up through complexes with 20+ subunits. This behavior is likely biologically important, as has been shown for other members of the S100 protein family. Further, we must know the oligomeric state of the protein under a given set of conditions to interpret our target- binding and spectroscopic studies. To complete the third aim of this grant, we must measure the oligomeric states of S100A5 and S100A6 in the presence of a large number of target proteins and under different solution conditions. A light scattering instrument will allow direct characterization of protein oligomeric state. This will have a high impact on our ability to complete Aim 3 of the parent grant by: 1) Allowing us to better understand the biochemistry and biology of these proteins, with specific reference to their oligomeric state and 2) Allowing us to interpret other functional and biochemical experiments, whose outcome depends strongly on the oligomeric state of the protein.
The goal of the parent grant is to experimentally and computationally identify new target proteins bound by the proteins S100A5 and S100A6. These proteins are highly upregulated in a variety of diseases including many cancers and heart disease; however, their binding targets?and therefore, biological functions?remain poorly described. We have found that these proteins can form large, multi-protein complexes in solution, complicating our ability to understand their biological targets and functions. The equipment supplement will be used to acquire an instrument that allows us to directly study the assembly of these complexes and thus better understand their biochemistry and function.
| Duvvuri, Hiranmayi; Wheeler, Lucas C; Harms, Michael J (2018) pytc: Open-Source Python Software for Global Analyses of Isothermal Titration Calorimetry Data. Biochemistry 57:2578-2583 |
| Wheeler, Lucas C; Anderson, Jeremy A; Morrison, Anneliese J et al. (2018) Conservation of Specificity in Two Low-Specificity Proteins. Biochemistry 57:684-695 |
| Wheeler, Lucas C; Harms, Michael J (2017) Human S100A5 binds Ca2+ and Cu2+ independently. BMC Biophys 10:8 |
