Structural And Intrinsic Disorder In The Regulation Of Protein-Protein Interactions

Abstract

This thesis applied spectroscopy and molecular dynamics simulation to study the structural biology of actin-binding domains (ABDs) from the spectrin superfamily of proteins as well as an intrinsically disordered region (IDR) of an integral membrane protein called synaptotagmin 1. In the former case, the structural hypothesis being tested was that actin-binding domains exist in distinct conformational states that are either permissive to or inhibitory towards binding of actin filaments. This question was probed using pulsed-EPR, which measured distances between the calponin homology (CH) domains that make up the ABD as proxy for conformation in the presence or absence of actin or with and without disease-causing mutation. The initial hypothesis of a closed compact state being unable to bind actin and an open extended state being binding-competent was largely supported by the data. However, the hypothesis was ultimately refined to conclude that an “open” state is likely to still be a fairly collapsed structure that is dynamically disordered. With this model, future efforts will be able use the model to look for small molecules that perturb the conformational equilibrium of ABDs harboring disease-causing mutations in potentially therapeutically efficacious ways. Moreover, the model can be tested in other ABDs of the protein superfamily to assess similarities and differences in mechanism. In the case of the intrinsically disordered region of synaptotagmin 1, it was hypothesized that a post-translational modification, specifically phosphorylation of a threonine residue, caused a structural change in the IDR that then results in a change in neurotransmitter release. This hypothesis was also tested with spectroscopic methods, mainly FRET and circular dichroism, but also with molecular dynamics. It was found that mimicking the low dielectric environment of the membrane with co-solvents in solution and artificially in silico caused the synaptotagmin 1 IDR to fold into helical structure. The post-translational modification, however, was found to interfere with the formation of helical structure, providing a still incomplete but novel molecular explanation for the effect it has on potentiation of neurotransmitter release observed in vivo. At the very least, the structural model provides a working hypothesis that can be further explored in further work.