Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.
© 2023 by the Authors.
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Original Publication Citation
Pandey, S., Raut, K. K., Clark, A. M., Baudin, A., Djemri, L., Libich, D. S., Ponniah, K., & Pascal, S. M. (2023). Enhancing the conformational stability of the cl-Par-4 tumor suppressor via site-directed mutagenesis. Biomolecules, 13(4), 1-15, Article 667. https://doi.org/10.3390/biom13040667
Pandey, Samjhana; Raut, Krishna K.; Clark, Andrea M.; Baudin, Antoine; Djemri, Lamya; Libich, David S.; Ponniah, Komala; and Pascal, Steven M., "Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis" (2023). Chemistry & Biochemistry Faculty Publications. 249.