The overarching goal of this program was to understand further the charged, disordered interactions that intrinsically disordered proteins (IDPs) can undergo.
We started by addressing the interaction of alpha-synuclein with the disordered loop of the plasma membrane calcium ATPase (PMCA). We found, using nuclear magnetic resonance (NMR) that the interaction between these two proteins was not direct, and therefore not governed by their charges. We then characterized the PMCA loop, which had previously been difficult due to its disorder. The manuscript describing this is currently in preparation.
Our next strategy for probing disordered protein interactions was to test the disorder-order continuum of interactions, carefully choosing a set of protein interacting pairs from being very disordered to ordered. For each pair, we made a peptide ligand binding partner from either L-amino acids (the left-handed, naturally occurring enantiomer) or D-amino acids (the right handed, rare enantiomer). Although having the same amino acid sequence, D-peptides are the mirror image of L-peptides and therefore will fold in the opposite direction to L-peptides. This makes the likelihood of their interactions with an L-protein binding partner very low, if structural rearrangement and adaption is necessary for the peptide to fit into a binding pocket. We hypothesized that this might not be true for proteins that remain disordered upon binding.
We first tested this using a disordered protein binding pair, finding that if there is no structure formed upon binding, a D-peptide can interact equally well as an L-peptide. Following this finding, we moved onto a protein pair in which the peptide is required to fold upon binding. In this case, the D-peptide could not interact with its binding partner, while the L-peptide interacted as expected. We therefore observed a clear distinction between a disordered protein interaction, versus one that required the formation of structure.
Following this, we investigated three different protein binding pairs, each with varying degrees of structure and disorder. We found that, instead of enantiomeric binding being black and white in disordered and ordered interactions, there were intermediate interactions that could occur depending on the extent of remaining disorder in the interaction.
In addition to this major finding, we also published a review on IDP phosphorylation and the implications of adding charges to an IDP post-translationally, and two research studies on charge distribution in IDPs and IDP size prediction.
Dissemination achievements:
• Three research papers published (The EMBO Journal, Biomolecules, Biophysical Journal)
• One review published (Essays in Biochemistry)
• One manuscript under review
• Three international conferences (one oral presentation: USA, two poster presentations: Australia, Switzerland)
