pH-Dependent Compaction of the Intrinsically Disordered Poly-E Motif in Titin

SIMPLE SUMMARY: The importance of pH shifts in biological systems is often underappreciated, but they can be a common form of regulation by both activating and inactivating important proteins. Proteins without any regular structure, commonly referred to as intrinsically disordered proteins, are very sensitive to pH changes due to their high number of ionizable amino acids. This study focuses on the role of non-ionizable residues in the negatively charged poly-E motif from the PEVK region of the muscle protein titin. Our results demonstrate that aromatic amino acids and the number of proline residues in the linker regions between charged clusters have the greatest impact on the pH-dependent conformational flexibility of this region. These results provide useful new insights into how the sequence between clusters of charges in proteins can impact the sensitivity of a protein to pH shifts. ABSTRACT: The conformational sensitivity of intrinsically disordered proteins to shifts in pH due to their high degree of charged residues has been recognized for well over a decade. However, the role of the non-ionizable residues in this pH sensitivity remains poorly understood. Our lab has been investigating the pH sensitivity of the poly-E motifs of the PEVK region of the muscle protein titin, which provides an ideal model system to explore this question. Using a series of 15-amino acid peptides derived from one of the poly-E motif sequences, we have investigated the role of side-chain chemistry in the conformational flexibility of this region. Our results demonstrate that aromatic side chains and proline content are the two variables that most influence pH sensitivity. The introduction of aromatic side chains resulted in a more collapsed structure, even at pH 7, while the removal of prolines resulted in a higher degree of pH sensitivity. These results highlight the importance of considering the impact of non-ionizable residues on IDP function, especially when considering the impact of pH on conformational flexibility.