A Functional Study of Plant Protein Villin4 and The Application of Sortase Mediated Ligation in Intrinsically Disordered…

Villin proteins are a family of filamentous actin (F-actin) regulators, playing vital roles in the organization of cytoskeletons in eukaryotes. Villin4, found in A. thaliana has a distinct structure that contains a folded C-terminal headpiece, a 192-residue disordered 'linker', and a folded N-terminal core domain. Villin4 has been shown to bind and bundle F-actin in the C-terminal headpiece domain. This disordered linker, or intrinsically disordered region (IDR), lacks the defined three-dimensional structure often found in proteins. IDRs are not novel. In fact, over 30% of eukaryotic proteins contain a sizable intrinsically disordered region over 50 amino acids in length. These IDRs are multifunctional and play a variety of crucial roles in the function of proteins. Because of the dynamic nature of IDRs, most standard biochemical methods of structural determination fall short. We have developed methods specifically suited to gain insight into proteins containing these IDRs, including segmentally labeled sortase mediated ligation, pull down assays and covalent modification studies. Through these efforts we have formulated a robust method of characterizing and optimizing the relative reactivity of sortase mediated ligation sites within authentic IDRs. We probed the backbone structure and dynamics of Villin4's linker using NMR, as well as calculating the specific and nonspecific binding capacity by formulating a holistic actin binding curve. We also probed predicted regions of the linker for specific covalent modifications in plant cell extract over a variety of time scales. Our work with sortase mediated ligation introduces a new method for researching IDRs. Using the knowledge gained in our covalent modification studies and our pull-down assays we lay the foundation for developing more robust crops.