Thursday, November 21, 2024
11/21/2024
Project Summary The primary goal of R01GM148610: “EmCAST: Stabilizing Proteins and Tuning Dynamics with High Precision and Accuracy” is to further develop and validate the EmCAST algorithm, which uses sequence/structure data available in the Protein Data Bank to predict stabilizing mutations at surface exposed sites of proteins. This goal will be accomplished in three aims. Aims 1 and 2 apply the method to four all- domains, two all- domains and a set of four / domains. Aim 3 applies EmCAST to the problem of tuning the equilibrium between two protein conformers. Reliable methods to stabilize proteins are essential for increasing the shelf life of protein pharmaceuticals and for providing stable scaffolds for evolving new protein functions and high- specificity binding agents. A key need of this project is the ability to efficiently express multiple variants of eleven different proteins from Escherichia coli. Recently, one of two incubator shakers in the PI’s lab has become less reliable leading to substantial downtime. This has significantly slowed the ability of the PI’s lab to express the proteins needed for this project. To remedy this problem, we are requesting funds through this equipment supplement to purchase an Eppendorf Innova 44R Incubator Shaker. This incubator shaker has refrigeration providing for tight temperature control in the 30 – 40 oC range (±0.1 oC) and incubation at temperatures up to 20 oC below ambient temperature. Shaking speeds can also be controlled to within ±1 rpm. These features will permit readily reproducible expression of the proteins needed for this project. The refrigeration capabilities of the Innova 44R are not available on our current incubator shaker and will facilitate expression of isotopically labeled proteins required to evaluate the effects of protein stability on dynamics in Aims 1 and 2 using NMR relaxation methods. Efficient production of isotopically labeled proteins on minimal media often requires incubation at ambient temperature or below. Finally, the robust and well-tested design of the Innova 44R ensures that the cost of its upkeep and maintenance will be minimal.
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