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The disordered PEVK region of titin contains two main structural motifs: PPAK and poly-E. The distribution of these motifs in the PEVK region contributes to the elastic properties of this region, but the specific mechanism of how these motifs work together remains unclear. Previous work from our lab has demonstrated that 28-amino acid peptides of the poly-E motif are sensitive to shifts in pH, becoming more flexible as the pH decreases. We extend this work to longer poly-E constructs, including constructs containing PPAK motifs. Our results demonstrate that longer poly-E motifs have a much larger range of pH sensitivity and that the inclusion of the PPAK motif reduces this sensitivity. We also demonstrate that binding calcium can increase the conformational flexibility of the poly-E motif, though the PPAK motif can block this calcium-dependent change. The data presented here suggest a model where PPAK and calcium can alter the stiffness of the poly-E motif by modulating the degree of charge repulsion in the glutamate clusters.

Regulation of Poly-E Motif Flexibility by pH, Ca2+ and the PPAK Motif