IA3 is an intrinsically disordered protein (IDP) from S. cerevisiae known for its inhibition of Aspartic proteinase A. The lack of secondary or tertiary structure in IDPs confers non-specificity of binding and thus allows each protein to be capable of several functions. IDPs are commonly composed of both positively and negatively charged amino acids and hence are polyampholytes. Fractional charge is a parameter that affects aspects such as hydration environment and protein structure. However, previous studies have shown that merely measuring the fraction of charged residues (FCR) to study these aspects is insufficient. Thus, the charge segregation (κ) of such systems can be used as an additional parameter to provide better information. Larger values of κ are associated with more clumped conformations whereas lower values are associated with well-mixed, or charge-balanced sequences, which are usually more extended conformations. In this project, we use site-directed mutagenesis to produce 6 IA3 mutations: N52E, E68N, K24L, L60K, D46K, and K61D. The mutations are paired in a manner that will change κ while leaving FCR constant. The effects of the changes in κ in the expressed proteins are studied via Electron Paramagnetic Spectroscopy and Circular Dichroism techniques. The utility of κ in predicting hydration environment and protein structure will be discussed in this work.