Towards the Flip of FruR to Er23 via Disulfide Bond Modification
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Date
2014-05
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The Ohio State University
Abstract
Understanding protein stability and the basis for why a protein folds are very important for the design of biological drugs for treatment of disease. Design of proteins has allowed researchers to discover and analyze many aspects of what it takes to make a protein fold and function under certain conditions, which is one of the key problems in biochemistry. For my project, we are working with two proteins, the DNA binding domain from fructose repressor (FruR or Cra), and a pheromone Er23. These proteins are good sources for mutation analysis because they are small and can be easily manipulated. This aspect will be useful for this project because our main goal is to engineer the FruR sequence to switch to the fold of Er23 or vice versa. We characterize these proteins using NMR, CD, crystallography, and fluorescence. Here we describe a successful variant of FruR that we created by making a mutation at position 25 from an alanine to a tryptophan. This tryptophan probe not only made the protein more thermodynamically stable by a factor of 5 °C, but will allow us to use high-throughput methods and fluorescence in order to observe it’s folding and unfolding much more efficiently. We have now turned our attention to expression of the wild type protein Er23, and preliminary results on expression of this pheromone will be described. We will also discuss a strategy to make the smallest number of mutations necessary for Er23 to adopt the FruR fold. We will do this by first engineering the cysteine residues out of Er23, replacing them with Ala-Val mutations. This is because Ala-Val is similar to a cysteine pair in size and we have also had success using this type of mutation with another protein the lab characterizes called Rop. If we are able to completely flip the fold of Er23 into FruR, this will give us knowledge of what it takes to make a protein fold and what conditions are needed to do so.
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Keywords
protein folding, Er23, FruR, disulfide bond modification, flipping fold