Investigating the Impact of Charge Patterning on Ensembles of IA3

Tianyan Li

University of Florida

IDPs and proteins containing an intrinsically disordered region (IDR) lack a well-defined tertiary structure and are a class of proteins that extend beyond the standard structure-to-function paradigm. A very small protein, IA3, is an endogenous inhibitor of the yeast aspartic proteinase YPrA. Although this inhibitor consists of 68 residues, its inhibitory activity resides within the N-terminal domain (NTD) half of the molecule, consisting only of residues 2–34, revealing an unexpected mode of inhibitor–enzyme interactions. We explored the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of YPrA by folding its NTD into an amphipathic alpha helix, while the C-terminal domain (CTD) remains unresolved. To better understand the charge patterning on protein ensembles, we conducted a secondary structure study on different strains of IA3 using Circular Dichroism Spectroscopy in the presence of 2,2,2-trifluoroethanol (TFE), a secondary structure inducer. In a previous hydration dynamics study, the C-terminal showed a more compact structure, whereas the N-terminal was more extended. To investigate the unresolved C-terminal, we separated the NTD and CTD as individual proteins. To check the helical propensity, we first applied a CD study on these separated NTDs and CTDs. Our preliminary experimental results suggest that the NTD tends towards an antiparallel structure in the absence of TFE. However, contrary to our hypothesis, TFE did not induce helix formation in the NTD. In some CTD samples, helix formation was observed in the presence of TFE. For purification purposes, the proteins were overexpressed with 6xHis-tags linked with a thrombin cleavage site. Therefore, optimization of the purification and thrombin cleavage process is necessarily required. Then, we will also look at whether the His-tag would impact the secondary structures of IA3s and their helical propensities.