Abstract 2169 Probing hidden structure within intrinsically disordered protein domains with chiral mutagenesis

Certain protein intrinsically disordered regions (IDRs) reversibly form higher-order oligomers to fulfill their cellular functions. As IDRs lack predictable structured elements, determination of sequence features that encode IDR oligomerization is an active area of study. In particular, the contribution of transiently-structured interactions to IDR oligomerization remains challenging to assess using conventional biochemical and biophysical techniques. To surmount this challenge, a recent study from our group utilized "backbone mutagenesis" wherein backbone amide hydrogen bond donors within IDR sequences were synthetically removed via Nα -methylation or ester bond substitution. This study indicated that position-specific backbone-dependent interactions contribute significantly to IDR oligomerization. The objective of our current study is to identify positions within IDRs that promote oligomerization through hidden ordered elements by utilizing a "chiral mutagenesis" strategy. This strategy involves substitution of L -enantiomer amino acids within an IDR sequence with their mirror-image non-proteogenic D-enantiomer counterparts using synthetic protein chemistry. "Chiral mutagenesis" through D-amino acid substitution within an L-amino acid protein disrupts local secondary structure due to unfavorable side chain geometries. Perturbation of IDR oligomerization through chiral mutagenesis therefore reveals sites within an IDR that apparently require ordered side chain or amide backbone geometries for activity. To produce heterochiral IDR constructs with L-to-D amino acid inversions at precisely defined positions, we utilize Expressed Protein Ligation to combine a recombinant protein fragment with a synthetic peptide bearing D-amino acid substitutions. The resultant semi-synthetic IDR proteins are assessed for self-association with reconstituted biochemical assays. Pulldown experiments employing chiral mutagenesis of the disordered nuclear lamina protein Emerin define a minimal amino acid window necessary for self-association through an enantioselective interaction. Additionally, chiral mutagenesis of intermediate filament proteins reveals that enantioselective interactions between disordered head domains are required for mature intermediate filament formation as indicated by negative-stain electron microscopy. In summary, we have developed a mutagenesis strategy to probe hidden secondary structures within IDRs using perturbation to amino acid site chain chirality rather than conventional side-chain mutagenesis. Using semi-synthetic heterochiral proteins, we show that hidden ordered elements in critical regions of IDRs drive self-association, and disruption of these elements inhibits physiological self-association. I would like to acknowledge funding from the Cancer Prevention and Research Institute of Texas (RR180051), the Welch Foundation (I-2039-20230405), the National Institute of General Medical Sciences (1R35GM147140), and the UT Southwestern Haberecht "Wildhare" and UT Southwestern Muscular Dystrophy Research Cooperative training grants.