Small Heat Shock Proteins (sHSPs) play crucial roles in protein homeostasis, the state of maintaining steady internal cellular conditions, despite changes to the cellular environment. As the “molecular life rafts” of the cell, sHSPs target partially misfolded proteins during stress measured as chaperone activity, and without consuming energy, prevent toxic aggregation. Mutations or malfunctioning of the sHSP, HSPB5 (B5), in humans are associated with neurodegenerative diseases such as Alzheimer's and Alexander's disease, as well as cancers, myopathies, and cataracts. As one of the ten sHSPs encoded in the human genome, B5 contains structural elements (the building block) common to all sHSPs: a highly conserved alpha-crystallin domain (ACD), that is flanked by variable, less conserved, N- and C-terminal regions (NTR and CTR, respectively). Previous studies determined that interactions between building blocks occur between a three amino acid region known as the I-X-I motif (a “knob”) in the CTR, and a hydrophobic groove (“hole”) in the neighboring block, similar to how two pieces of Lego come together to build a larger structure. Interestingly, in B5, there is an additional I-X-I motif in the unfolded NTR, but its role and structure are unknown. I hypothesize that the CTR and NTR motifs compete for binding into the hydrophobic groove, indicating the “knob” into “hole” interaction is loose, where the other “knob” can bind the “hole” when one leaves. To test this hypothesis, I prepared mutants containing CTR, NTR, and double motif deletions. I aim to uncover the effects of these mutations on chaperone activity and oligomeric size, by optimizing protein purification and using a combination of in vitro biochemical assays, and native (non-denaturing) gel electrophoresis on purified B5 mutants. These results will assist in demystifying the role of the NTR, direct future sHSP studies, and provide important insight for development of future therapeutic strategies.